PT2185198E - Lox and l0xl2 inhibitors and uses thereof - Google Patents

Description

DESCRIPTION

BACKGROUND OF THE INVENTION Cancer is a serious public health problem in the United States and other developed countries. Currently, one in four deaths in the United States is due to cancer. Cancer therapy involves treating patients with chemotherapeutic drugs to kill tumor cells. However, tumor cell subsets are often resistant to drug therapy and survive to repopulate at sites of origin and at distant metastatic sites, leading to recurrence and morbidity of detectable disease. Many carcinoma tumor cells which have the properties of increased mastastatic and invasive capacity, and altered drug resistance, are thought to undergo a morphological transformation encompassing or similar to EMT (epithelial-mesenchymal transition). Cells subjected to EMT lose the normal adhesive properties of epithelial cells and undergo a spectrum of changes, including loss of E-cadherin expression and expression of mesenchymal markers, increased motility, increased invasiveness, and resistance to increased cell death.

The leading therapies for cancer are currently surgery, radiation and chemotherapy. Chemotherapeutic approaches, such as antitumor antibiotics, alkylating agents, nitrosourea compounds, vinca alkaloids, steroid hormones and anti-metabolites form the majority of the therapeutics available to oncologists. Despite advances in the field of cancer treatment, cancer remains a major health problem.

Angiogenesis, the formation of new blood vessels outside the pre-existing capillaries, is a sequence of events that is of crucial importance in the wide range of physiological and pathological processes. Normal tissue growth, such as in embryonic development, healing and menstrual cycle, is characterized by the dependence on the formation of new vessels for the supply of oxygen and nutrients, as well as removal of waste products. A large number of different and unrelated diseases are also associated with the formation of new vasculature. Among certain pathologies are pathological conditions in which angiogenesis is low and should be potentiated to ameliorate disease conditions. More often, however, excessive angiogenesis is an important feature of various pathologies, including pathologies characterized or associated with abnormal or uncontrolled proliferation of cells. Pathologies involving excessive angiogenesis include, for example, cancer (both solid and haematological tumors), cardiovascular diseases (such as atherosclerosis and restenosis), chronic inflammation (rheumatoid arthritis, Crohn's disease), diabetes (diabetic retinopathy), psoriasis, endometriosis, neovascular glaucoma and adiposity (3). These pathological conditions may benefit from chemotherapeutic inhibition of angiogenesis.

Generally speaking, the angiogenic process requires the proliferation and migration of a normally quiescent endothelium, the controlled proteolysis of the pericellular matrix and the synthesis of new components of the extracellular matrix developing capillaries. The establishment of new intra- and intercellular contacts and the morphological differentiation of endothelial cells into capillary-type tubular networks provide support for their subsequent maturation, branching, remodeling and selective regression to form a highly organized, functional microvascular network. The autocrine, paracrine, and amphrine interactions of the vascular endothelium with its surrounding stromal components, as well as pro-angiogenic and angiostatic cytokines and growth factors orchestrating physiological angiogenesis, are usually tightly regulated both spatially and temporally. Angiogenesis is crucial for the growth of neoplastic tissues. For more than 100 years, tumors were observed to be more vascular tissues than normal. Several experimental studies have suggested that both primary tumor growth and metastasis require neovascularization. In contrast to the well-orchestrated process described above for normal tissue growth, pathological angiogenesis required for active tumor growth is generally prolonged and persistent, with the initial acquisition of the angiogenic phenotype being a common mechanism for the development of a variety of solid and hematopoietic tumor types. Tumors that are unable to recruit and sustain a vascular network typically remain dormant as asymptomatic lesions in situ. Metastasis is also dependent on angiogenesis: for a metastatic tumor cell to succeed, it usually has to gain access to the vasculature in the primary tumor, survive the circulation, halt the microvasculature of the target organ, exit this vasculature, grow in the target organ, and induce angiogenesis at the target site. Thus, angiogenesis seems to be necessary at baseline, as well as the completion of the metastatic cascade. The criticality of angiogenesis for the growth and metastasis of neoplasms thus provides a potentially optimal target for chemotherapeutic efforts. Appropriate anti-angiogenic agents may act directly or indirectly to influence tumor associated angiogenesis by delaying its onset (i.e., blocking an " angiogenic intervener ") or by blocking prolonged and focal neovascularization which is characteristic of many tumor types. Anti-angiogenesis therapies directed against tumor associated endothelium and the multiple molecular and cellular processes and targets implicated in prolonged pathological angiogenesis are being actively evaluated for their safety and efficacy in multiple clinical trials. However, there has been limited success to date in the discovery and / or identification of safe and / or effective anti-angiogenic agents.

Fibrosis is the abnormal accumulation of fibrous tissue that can occur as a part of the healing process in damaged tissue. Such tissue damage can result from physical injury, inflammation, infection, exposure to toxins, and other causes. Liver (hepatic) fibrosis, for example, occurs as a part of the healing response to chronic liver injury. Fibrosis occurs as a complication of hemochromatosis, Wilson's disease, alcoholism, schistosomiasis, viral hepatitis, obstruction of the bile duct, exposure to toxins, and metabolic disorders. This healing tissue formation is thought to represent an attempt by the body to encapsulate the injured tissue. Liver fibrosis is characterized by the accumulation of extracellular matrix that can be distinguished qualitatively from that of the normal liver. If left untreated, hepatic fibrosis progresses to cirrhosis (defined by the presence of encapsulated nodules), liver failure, and death.

As summarized by Li and Friedman (Gastroenterol, Hepatol., 14: 618-633, 1999), proposed and actual therapeutic strategies for hepatic fibrosis include removal of the underlying cause (e.g., toxin or infectious agent), suppression of inflammation , for example, corticosteroids, IL-1 receptor antagonists or other agents), down regulation of the activation of star cells using, for example, gamma interferon or antioxidants), promoting matrix degradation or promoting apoptosis of star cells. Despite recent progress, many of these strategies are still in the experimental phase and existing therapies aim to suppress inflammation rather than target the underlying biochemical processes. Thus, there remains a need in the art for materials and methods for treating fibrosis, including pulmonary and hepatic fibrosis.

Fibrotic tissues accumulate in the heart and blood vessels as a result of hypertension, hypertensive heart disease, atherosclerosis, and acute myocardial infarction. High blood pressure, or hypertension, can be caused by a variety of factors and often leads to the development of hypertensive heart disease (HHD) with progression to cardiac arrest and acute myocardial infarction. Likewise, atherosclerosis and other ischemic heart diseases also often result in cardiac arrest. These cardiovascular diseases all exhibit an accumulation of extracellular matrix or fibrotic deposition which results in the stiffening of the vasculature and stiffening of the cardiac tissue itself. This deposition of fibrotic material is a response to damage induced by the hypertensive and / or sclerotic state, but the effects of this response also result in the negative effects of vascular and cardiac tightening as well as increased ventricle. In addition, increased cardiac fibrosis observed in cardiovascular disease is thought to interrupt or alter the signals transmitted to cardiomyocytes through scaffolding of cardiac tissue, further leading to disruption of efficient cardiac function and promoting cardiac arrest and acute myocardial infarction. WO 00/44910 describes polypeptides of proteins and Lor-2 antibodies. Peinado et al, EMBO Journal, vol. 24, No. 19, 2005, pages 3446-3458; and Peinado et al., Cancer Research 2008, vol. 68, No. 12, 15 pages 4541-4550, relates to LOXL2 and its use as a prognostic marker for squamous cell carcinomas. US 2007/021365 relates to methods of identifying LOX inhibitors and the use of such inhibitors to prevent or treat tumors.

SUMMARY OF THE INVENTION

Epithelial-to-Mesenchymal Transition (EMT) refers to the process by which a cell with a characteristic gene expression / epithelial cell (i.e., expressing specific proteins, factors, and molecules) changes or alters the genes or their levels expression resulting in a change in the phenotype of the cell as exhibited by the change or change in the expressed genes. EMT-preventing compositions are required and are effective in blocking LOXL2 activity. Such inhibitors are useful in the treatment of diseases and disorders associated with aberrant levels of LOXL2. The present invention provides an isolated antibody or antigen-binding fragment thereof which binds to a lysyl oxidase-2 (LOXL2) protein, wherein said antibody or antigen-binding fragment binds specifically to the amino acid sequence of SEQ. ID No. 6 and inhibits the enzymatic activity of the LOXL2 protein, in which the inhibition is non-competitive. These and other embodiments of the invention are set forth in the description and the appended claims. Reference herein to LOXL2 antibodies means antibodies of the invention, as defined above.

Antibodies that bind to enzymes may be competitive inhibitors, competitive inhibitors or noncompetitive inhibitors. In relation to competitive inhibition, an inhibitor normally has structural similarity to the substrate. Inhibition will be evident at low substrate concentrations, but may be overcome at elevated substrate concentrations. With respect to the inhibitory activity, an inhibitor binds to a site which becomes available after the substrate binds to the active site. Inhibition will be more evident at elevated substrate concentrations. With respect to noncompetitive inhibition, an inhibitor binds to a site away from the substrate binding site and the relative inhibition will generally be the same at all substrate concentrations. In one embodiment, an antibody or antigen-binding fragment thereof, described herein, specifically binds to both full-length and processed L0XL.2. In one aspect, both full-length and processed L0XL2 are active forms of the enzyme.

In one aspect, the L0XL2 antibody of the invention that binds to the amino acid of SEQ. ID No. 6 comprises a variable heavy chain having at least 75% amino acid sequence identity with an amino acid sequence set forth as SEQ. ID No. 1 and a variable light chain with at least 75% amino acid sequence identity to an amino acid sequence set forth as SEQ. ID No. 2.

In one embodiment, an isolated L0XL2 antibody or antigen-binding fragment thereof comprises a variable heavy chain having at least 75% amino acid sequence identity with an amino acid sequence shown as SEQ. In another embodiment, an isolated L0XL2 antibody or antigen-binding fragment thereof comprises a variable light chain with at least 75% amino acid sequence identity to an amino acid sequence set forth as SEQ. ID No. 2. In yet another embodiment, an isolated L0XL2 antibody or antigen-binding fragment thereof competes with or specifically binds to any of the anti-L0XL2 antibodies or antigen-binding fragments thereof as described herein for connection to L0XL2. Antibodies or antigen-binding fragments thereof may bind specifically to L0XL2 with a binding affinity of at least 2, 5, 10, 100, 100, 500 or 1000-fold greater than at least one of LOX, LOXL1, LOXL3 or LOXL4. Humanized anti-LOXL2 antibodies are provided herein. In one embodiment, the humanized LOXL2 antibody or antigen-binding fragment thereof comprises a variable heavy chain having at least 75% amino acid sequence identity with an amino acid sequence shown as SEQ. 25, 26, 27 or 28 and a variable light chain with at least 75% amino acid sequence identity to an amino acid sequence set forth as SEQ. ID No. 30, 31 or 32.

An isolated humanized LOXL2 antibody or antigen-binding fragment thereof may comprise a variable heavy chain with at least 75% amino acid sequence identity to an amino acid sequence shown as SEQ. 25, 26, 27 or 28 and a variable light chain with at least 75% amino acid sequence identity to an amino acid sequence set forth as SEQ. ID No. 30, 31 or 32.

A humanized LOXL2 antibody or antigen-binding fragment thereof may comprise a variable heavy chain with at least 75% amino acid sequence identity to an amino acid sequence set forth as SEQ. 25, 26, 27 or 28.

A humanized LOXL2 antibody or antigen-binding fragment thereof may comprise a variable light chain with at least 75% amino acid sequence identity to an amino acid sequence set forth as SEQ. ID No. 30, 31 or 32. Combinations of variable heavy chains and variable light chains can be made to assess binding affinity. There is provided herein a humanized LOXL2 antibody or antigen-binding fragment thereof, which competes with or specifically binds to an antibody or antigen-binding fragment thereof as described herein for binding to L0XL2. There is provided herein a humanized L0XL2 antibody, or antigen-binding fragment thereof, which binds to L0XL2, comprising a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region comprises: ) a heavy chain FR1 with the amino acid sequence of SEQ. ID No. 33 or the amino acid sequence of SEQ. ID No. 33, but by one or more substitutions selected from the group consisting of: (a) a glutamine (Q) substitution by valine (V) or a conservative substitution thereof at position 24; (b) a leucine (L) substitution per valine (V) or a conservative substitution thereof at position 30; (c) a substitution of valine (V) for lysine (K) or a conservative substitution thereof at position 31; (d) replacing arginine (R) with lysine (K) or a conservative substitution thereof at position 32; and (e) a substitution of threonine (T) for alanine (A) or a conservative substitution thereof at position 35; and a deletion of amino acid residues 1-19; (ii) a heavy chain FR2 with the amino acid sequence of SEQ. ID No. 34 or the amino acid sequence of SEQ. ID No. 34, but by one or more substitutions selected from the group consisting of: (a) a lysine (K) substitution by arginine (R) or a conservative substitution thereof at the 3-position; (b) an arginine (R) substitution by alanine (A) or a conservative substitution thereof at the 5-position, and (iii) a FR3 heavy chain having the amino acid sequence of SEQ. ID No. 35 or the amino acid sequence of SEQ. ID No. 35, but by one or more substitutions selected from the group consisting of: (a) a lysine (K) substitution by arginine (R) or a conservative substitution thereof at the 1-position; (b) a substitution of alanine (A) for valine (V) or a conservative substitution thereof at the 2-position; (c) a replacement of leucine (L) by isoleucine (I) or a conservative substitution thereof at the 4-position; (d) a serine (S) substitution for threonine (T) or a conservative substitution thereof at the 10-position; (e) a glutamine (Q) substitution by glutamic acid (E) or a conservative substitution thereof at position 16; (f) a replacement of threonine (T) by arginine (R) or a conservative substitution thereof at position 21; (g) replacing aspartic acid (D) with glutamic acid (E) or a conservative substitution thereof at position 23; (h) a serine (S) substitution for threonine (T) or a conservative substitution thereof at position 25; and (i) a substitution of phenylalanine (F) for tyrosine (Y) or a conservative substitution thereof at position 29; and (iv) a FR4 heavy chain having the amino acid sequence of SEQ. ID No. 36 or the amino acid sequence of SEQ. 36, but by a lysine (K) substitution for valine (V) or a conservative substitution thereof at position 7, and wherein said light chain variable region comprises: (i) a light chain FR1 with amino acid sequence of SEQ. ID No. 49 or the amino acid sequence of SEQ. ID No. 49, but by one or more substitutions selected from the group consisting of: (a) a substitution of alanine (A) with threonine (T) or a conservative substitution thereof at position 27; (b) a substitution of alanine (A) for proline (P) or a conservative substitution thereof at position 28; (c) a proline substitution (P) for leucine (L) or a conservative substitution thereof at position 29; (d) a substitution of valine (V) for leucine (L) or a conservative substitution thereof at position 31; (e) a substitution of glutamic acid (E) for glutamine (Q) or a conservative substitution thereof at position 37; (d) a substitution of serine (S) by proline (P) or a conservative substitution thereof at position 38; (f) a substitution of valine (V) for alanine (A) or a conservative substitution thereof at position 39; and a deletion of amino acid residues 1-20; (ii) an FR2 light chain with the amino acid sequence of SEQ. ID No. 50 or the amino acid sequence of SEQ. (A) a phenylalanine (F) substitution by tyrosine (Y) or a conservative substitution thereof in the 2-position; and (b) a substitution of arginine (R) for lysine (K) or a conservative substitution thereof at position 5; (iii) an FR3 light chain with the amino acid sequence of SEQ. ID No. 51 or the amino acid sequence of SEQ. ID 51, but by one or more substitutions selected from the group consisting of: (a) a substitution of alanine (A) with aspartic acid (D) or a conservative substitution thereof at the 14-position; and (b) a substitution of arginine (R) for lysine (K) or a conservative substitution thereof at position 18; and (iv) an FR4 light chain with the amino acid sequence of SEQ. ID No. 52 or the amino acid sequence of SEQ. ID No. 52, but by a leucine (L) substitution for valine (V) or a conservative substitution thereof at the 7-position;

In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a FR1 heavy chain variable region with an amino acid sequence as set forth in SEQ. ID No. 33, 37 or 44; a heavy chain variable region FR2 with an amino acid sequence as set forth in SEQ. ID No. 34, 38 or

45; a FR3 heavy chain variable region with an amino acid sequence as set forth in SEQ. ID No. 35, 39, 46, 47 or 48; a heavy chain variable region FR4 with an amino acid sequence as set forth in SEQ. ID No. 36 or 40; a FR1 light chain variable region with an amino acid sequence as set forth in SEQ. ID No. 49 or 53; a variable region of the FR2 light chain with an amino acid sequence as set forth in SEQ. ID No. 50, 54 or 60; an FR3 light chain variable region with an amino acid sequence as set forth in SEQ. ID No. 51, 55 or

61; and a FR4 light chain variable region with an amino acid sequence as set forth in SEQ. ID No. 52 or 59.

An isolated antibody or antigen-binding fragment thereof may be labeled with a detectable label, a therapeutic label, or both.

In one embodiment, an antigen binding fragment is, for example, a variable heavy chain, a variable light chain, a Fv, a scFv, a Fab, an F (ab ') 2, a genetically engineered antibody, an antibody monoclonal or humanized antibody. There is provided herein a kit for treating a pathological condition associated with LOXL2, which comprises a composition of an antibody or antigen-binding fragment thereof of any of the foregoing modalities and a pharmaceutically acceptable carrier or excipient. A pathological condition associated with L0XL2 may be, for example, a tumor, a metastasis, angiogenesis or fibrosis. A kit may further comprise a detectable label, a therapeutic label, or both. Kits may further comprise written instructions describing how to conjugate the antibody or antigen-binding fragment thereof to the detectable label, a therapeutic label, or both. In addition, written instructions may describe how to administer the antibody or antigen-binding fragment thereof. In one embodiment, the compositions in the kit are pyrogen-free and may, on some occasions, be lyophilized. There is provided herein a method of diagnosing a pathological condition associated with L0XL2 which comprises evaluating a level of L0XL2 in a sample of a subject contacting said sample with an antigen-binding antibody or fragment thereof as described herein, wherein a change in the level of L0XL2 in the sample compared to a reference sample indicates the presence or increase of a tumor or metastasis. A pathological condition associated with L0XL2 may be, for example, a tumor, a metastasis, angiogenesis or a fibrotic pathological condition. In one embodiment, an increase in L0XL2 levels in the sample compared to a reference sample indicates the presence of a tumor or metastasis or an increase in tumor or metastatic growth. A reference sample is a sample taken from the subject at an earlier time point or a sample from another subject. The levels of L0XL2 levels in the sample are detected by contacting the sample with any of the antibodies or antigen-binding fragments thereof as described herein. For the purpose of detection, an antibody or antigen-binding fragment thereof is detectably labeled as needed, depending on the method used to evaluate binding. There is provided herein a method of inhibiting L0XL2 by contacting a sample or a cellular tissue with an antibody or antigen-binding fragment thereof, described herein. In one embodiment, binding of said antibody or antigen-binding fragment thereof to L0XL2 inhibits the enzymatic activity of L0XL2.

Inhibiting LOX or L0XL2 may reduce tumor growth in a subject either partially or completely. Inhibiting L0XL2 may reduce angiogenesis in a subject such that a therapeutic benefit occurs. Inhibiting L0XL2 may reduce fibrosis in a subject, such that a therapeutic benefit occurs. There is provided herein a method of reducing the growth of a tumor in a subject, comprising administering an antibody or antigen-binding fragment thereof, as described herein. A tumor may be a primary tumor or a metastatic tumor. In one aspect, a tumor is, for example, lung cancer (including lung adenocarcinoma, squamous cell carcinoma, large cell carcinoma, bronchioloalveolar carcinoma, non-small cell carcinoma, small cell carcinoma, mesothelioma); breast cancer (including carcinoma, lobular carcinoma, inflammatory breast cancer, clear breast cancer, mucinous carcinoma); colorectal cancer (colon cancer, rectal cancer); anal cancer; pancreatic cancer (including pancreatic adenocarcinoma, islet cell carcinoma, neuroendocrine tumors); prostate cancer; ovarian carcinoma (ovarian epithelial carcinoma or tumor of the epithelial surface-stroma including serous tumor, endometrioid tumor and mucinous cystadenocarcinoma, sex-stromal tumor); hepatic and bile duct carcinoma (including hepatocellular carcinoma, cholangiocarcinoma, hemangioma); oesophageal carcinoma (including esophageal adenocarcinoma and squamous cell carcinoma); non-Hodgkin's lymphoma; bladder carcinoma; uterine carcinoma (including endometrial adenocarcinoma, uterine papillary serous carcinoma, uterine clear cell carcinoma, uterine sarcomas and leiomyosarcomas, mixed mulerian tumors); glioma, glioblastoma, medulablastoma and other brain tumors; renal cancers (including renal cell carcinoma, clear breast cancer, Wilm's tumor); cancer of the head and neck (including squamous cell carcinomas); stomach cancer (stomach adenocarcinoma, gastrointestinal stromal tumor); multiple myeloma; testicular cancer; germ cell tumor; neuroendocrine tumor; cervical cancer; carcinoids of the gastrointestinal tract, breast and other organs; signaling ring cell carcinoma; mesenchymal tumors including sarcomas, fibrosarcomas, hemangioma, angiomatosis, hemangiopericytoma, pseudoangiomatous stromal hyperplasia, myofibroblastoma, fibromatosis, inflammatory myofibroblastic tumor, lipoma, angiolipoma, granular cell tumor, neurofibroma, schwannoma, angiosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, leiomyoma or leiomyosarcoma . In one embodiment, a tumor is, for example, a tumor of the colon, an ovarian tumor, a lung tumor, an esophageal tumor, a mammary tumor, a prostate tumor, a carcinoma. The size of the tumor in the subject can be reduced by at least 10%, 25%, 50%, 70%, 90%, 95% or more after treatment when compared to the tumor in the subject prior to treatment. In one aspect, the survival of a subject with a tumor is increased by at least 10 days, 1 month, 3 months, 6 months, 1 year, 2 years, 5 years, 10 years or more compared to that of a subject to whom the antibody or antigen-binding fragment thereof is administered. The metastatic tumor burden of a subject can be stabilized following the administration of an antibody or antigen-binding fragment thereof, described herein. For example, the burden of metastatic tumors can be stabilized for at least 10 days, 1 month, 3 months, 6 months, 1 year, 2 years, 5 years, 10 years or more. There is provided herein a method of inhibiting angiogenesis in a subject by an antibody or antigen-binding fragment thereof as described herein. There is provided herein a method of inhibiting a fibrotic disease in a subject by administering an antibody or antigen-binding fragment thereof as described herein. Fibrotic diseases include, but are not limited to, hepatic fibrosis, pulmonary fibrosis, renal fibrosis, cardiac fibrosis, and scleroderma. In one embodiment, renal fibrosis includes, but is not limited to, diabetic nephropathy, vesicoureteral reflux, tubulointerstitial renal fibrosis; glomerulonephritis or glomerular nephritis, including focal segmental glomerulosclerosis and membranous glomerulonephritis and mesangiocapillar glomerular nephritis. In one embodiment, hepatic fibrosis results in cirrhosis, and in associated pathological conditions such as chronic viral hepatitis, non-alcoholic fatty liver disease (NAFLD), alcoholic steatohepatitis (ASH), non-alcoholic steatohepatitis (NASH), biliary cirrhosis (PBC), biliary cirrhosis and autoimmune hepatitis. There is provided herein a method of decreasing the formation of extracellular matrix by contacting a sample or tissue with an antibody or antigen-binding fragment thereof as described herein. Administration or contact may occur, for example, by parenteral administration. There is provided herein a method of monitoring the response of a subject to the administration of an antibody or antigen-binding fragment thereof, described herein by detecting the levels and / or activity of LOXI2.

In one embodiment, said antibody or antigen-binding fragment thereof is labeled with a therapeutic label. Combination therapy is contemplated herein in which the methods further comprise co-administering a second therapeutic agent. In one embodiment, the second therapeutic agent is an antibody or a chemotherapeutic agent. There is provided herein a use of an antibody or antigen-binding fragment thereof, described herein in the preparation of a formulation for inhibiting L0XL2, reducing tumor growth, inhibiting angiogenesis, inhibiting fibrotic disease, or decreasing the formation of extracellular matrix in a subject . In one embodiment, said antibody or antigen-binding fragment thereof is labeled with a therapeutic label and, optionally, a diagnostic label. There is provided herein a use of an antibody or antigen-binding fragment thereof, described herein in the preparation of a formulation for diagnosing a tumor or metastasis which comprises evaluating L0XL2 levels in a sample of a patient, wherein a change in levels of L0XL2 in the sample compared to a reference sample indicates the presence of a tumor or metastasis or an increase in tumor or metastatic growth. In one embodiment, said antibody or antigen-binding fragment thereof is labeled with a diagnostic label.

BRIEF DESCRIPTION OF THE DRAWINGS

Features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description which provides illustrative embodiments in which the principles of the invention are used and the accompanying drawings. Figure 1 shows the Enzyme of Lisil Oxidase. LOX / L enzymes act through a ping-pong mechanism that can be described by Michaelis-Menten kinetics. Figure 2 illustrates common modes of enzymatic inhibition. Figure 3 illustrates βΑΡΝ is a competitive inhibitor of L0XL2. Figure 4 illustrates modes of enzymatic inhibition: L0XL2. Figure 5 illustrates the location of extracellular L0XL2 and function of extracellular L0XL2.

Figure 6A provides the amino acid sequences of a heavy chain variable region and Figure 6B provides the amino acid sequences of a light chain variable region of an antibody that binds to the SRX3-4 region of L0XL2. For each variable region, peptides are shown italicized signals, CDRs are underlined and the beginning of the constant structure is shown in bold. Figure 7A provides the amino acid sequences of a heavy chain variable region and Figures 7B and 7C provide amino acid sequences of two light chain variable regions of antibodies that bind to LOX. For each variable region, peptides are shown italicized signals and CDRs are underlined. Figure 8 provides an update of protein B screening using anti-L0XL2 antibodies. The enzymatic activity of L0XL2 was evaluated. Figure 9 shows the enzymatic activity of the anti-L0XL2 antibody AB0023. Figure 10 demonstrates that the anti-LOXL2 antibody AB0023 is a non-competitive inhibitor. Figure 11 shows the binding affinity and the dissociation rate of the anti-LOXL2 antibody AB0023. Figure 12 shows the mapping of the anti-LOXL2 antibody domain AB0023; ο AB0023 binds to the SRCR domain 3-4 of LOXL2. Figure 13 shows that the anti-LOXL2 antibody AB0023 demonstrates inhibition of migration / invasion consistent with collagen I and collagen IV from supernatants through 10 ml of preparation material and 100 ml of extended preparation and ascites material. Partial inhibition was also observed in cell adhesion assays. In test samples, the cells in the assay migrate towards the serum and the fluorescence is measured to determine cell counting and migration. The leftmost bar is a control sample in which the antibody is present and the low layer contains serum (positive control for cell invasion). The second left bar is a negative control in which the antibody is present and the downstream layer does not contain serum. Figure 14 provides variable amino acid (VH) and variable light chain (VL) amino acid sequences of murine monoclonal antibody AB0023 (anti-hLOXL2). Complementarity-determining regions (CDRs) are shown in bold underline. Figure 14 also provides four humanized variants of the murine monoclonal antibody. Residues in the framework regions (FR) of the humanized variable light and heavy chains that differ from the murine monoclonal antibody are shown by interrupted (---) or italic underlining. Figure 15 demonstrates that M64 binds to LOX in a dose-dependent manner. Lot 3 has a KD of 6.6 nM, Lot 4 has a KD of 5.0 nM and Lot 5 has a KD of 5.7 nM. Figure 16 illustrates the binding affinity of the anti-LOX M64 antibody. Figure 17 demonstrates that anti-LOXL2 antibodies inhibited the cell growth of four cancer derived cell lines.

Figures 18 demonstrate a synergistic effect of an anti-LOX antibody in combination with cisplatin. IC50 values of M64 were also determined in four cell lines.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the field of medicine, including diagnosis and treatment of cancer. One aspect of the invention relates to L0XL2 as indicators of disease progression and a target for therapeutic agents. The present invention provides novel methodology and related composition and kits for diagnosing or monitoring various diseases associated with abnormal cell proliferation, angiogenesis and fibrosis, using agents which specifically recognize active or mature forms of lysyl oxidase (L0XL2) type proteins. Methods are provided for diagnosing or monitoring carcinogenic metastasis in a subject, comprising: assessing levels of active L0XL2 or activity in blood or a tumor by the subjects a change in active L0XL2 levels or activity in blood or tumor compared to a reference sample , indicates the presence of metastatic tumor growth.

As described in more detail below, active L0XL2 levels can be assessed by various methods including, but not limited to, immunohistochemistry using antibodies that bind specifically to the active or mature form of L0XL2. The enzymatic activity of the active L0XL2 can be measured using various methods including, but not limited to, fluorometric and chromogenic assays.

Also provided herein are antibodies or antigen-binding fragments thereof, which specifically recognize L0XL2 active forms, methods for generating antibodies against L0XL2 active forms, and method of using the antibodies to treat abnormal cell proliferation, angiogenesis and fibrosis. I. General Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. If a definition presented in this section is contrary to, or otherwise inconsistent with, a definition in patents, applications, published applications and other publications, the definition in this section prevails. Headers are provided here for convenience only.

As used herein, " one " or " one " means " at least one " or " one or more. " The phrase " conservative amino acid substitution " refers to the grouping of amino acids based on certain common properties. A functional way of defining common properties among individual amino acids is to analyze the standard frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, G. E. and R. H. Schirmer, Principles of Protein Structure, Springer-Verlag). According to such analyzes, amino acid groups can be defined where amino acids within a group preferentially exchange with each other and thus are similar to each other in their impact on the overall protein structure (Schulz, GE and RH Schirmer, Principles of Protein Structure, Springer-Verlag). Examples of amino acid groups defined in this way include: (i) a charged group consisting of Glu and Asp, Lys, Arg and His, (ii) a positively charged group consisting of Lys, Arg and His, (iii) a (iv) an aromatic group consisting of Phe, Tyr and Trp, (v) a nitrogen ring, consisting of His and Trp, (vi) a large non-polar aliphatic group consisting of Val, Leu and Ile, (vii) a slightly polar group, consisting of Met and

Cys, (viii) a small group of residues consisting of Ser, Thr, Asp, Asn, Gly, Ala, Glu, Gin and Pro, (ix) an aliphatic group consisting of Val, Leu, Ile, Met and Cys, and (x) a small hydroxyl group consisting of Ser and Thr.

In addition to the groups set forth above, each amino acid residue may form a group and the group formed by an individual amino acid may be referred to by a single and / or three letter abbreviation for that amino acid commonly used in the art, as described above.

A " conserved residue " is an amino acid that is relatively invariant over a range of similar proteins. Often, conserved residues will only change upon being replaced with a similar amino acid, as described above for " conservative amino acid substitution ".

It is intended that the letter " x " or " xaa ", as used in the amino acid sequences herein, indicate that any of the twenty standard amino acids may be placed in this position, unless specifically indicated otherwise. For the purposes of peptide-mimetic cleavage, an " x " or a " xaa " in an amino acid sequence may be substituted by a mimic of the amino acid present in the target sequence or the amino acid may be replaced by a spacer of essentially any form that does not interfere with the activity of the peptide-mimetic. " Homology " or " identity " or " similarity " refers to the similarity of sequences within two peptides or between two nucleic acid molecules. Homology and identity can each be determined by comparing one position in each sequence that can be aligned for comparison purposes. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position; when the equivalent site occupied by the same or a similar amino acid residue (for example, similar in the steric and / or electronic nature), then the molecules may be referred to homologous (like) in that position. Expression as a percentage of homology / similarity or identity refers to a function of the number of identical or similar amino acids at the positions shared by the compared sequences. A sequence that is " unrelated " or " non-homologous " shares less than 40% identity, although preferably less than 25% identity with a sequence of the present invention. By comparing two sequences, the absence of residues (amino acids or nucleic acids) or the presence of extra residues also decreases identity and homology / similarity. The term " homology " describes a mathematically ablative comparison of sequence similarities that is used to identify genes or proteins with similar functions or motifs. The nucleic acid (nucleotide, oligonucleotide) and amino acid (protein) sequences of the present invention may be used as a " query sequence " to conduct a search against public databases for, for example, identifying other members of the family, related or homologous sequences. Such searches can be performed using the NBLAST and XBLAST (version 2.0) programs of Altschul, et al. (1990) J. Mol. Biol. 215: 403-10. BLAST nucleotide searches can be performed with the NBLAST program, rank = 100, word length = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST amino acid searches can be performed with the XBLAST program, rank = 50, word length = 3 to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain alignments with intervals for comparative purposes, BLAST can be used with intervals as described in Altschul et al. , (1997) Nucleic Acid Res. 25 (17): 3389-3402. When using the BLAST and BLAST programs with intervals, the default parameters of the respective programs (for example, XBLAST and BLAST) can be used (see, www.ncbi.nlm.nih.gov).

As used herein, " identity " means the percentage of nucleotides or amino acid residues identical at the corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching, i.e., taking into consideration ranges and insertions. The identity can readily be calculated by known methods, including but not limited to those described in Computational Molecular Biology, Lesk, A.M., editor, Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods for determining identity are designed to give the highest match between the sequences tested. In addition, methods for determining identity are encoded in publicly available computer programs. Methods of computer programs for determining identity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acid Research 12 (1): 387 (1984)), BLASTP , BLASTN and FASTA (Altschul, SF et al., J. Molec Biol., 215: 403-410 (1990) and Altschul et al., Nuc. Acids Res. 25: 3389-3402 (1997)). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. The term " substantially identical " means identity between a first amino acid sequence that contains a sufficient or minimum number of residues of the same or different amino acid residues. amino acids which are i) identical to or ii) conservative amino acid residue substitutions aligned in a second amino acid sequence, so that the first and second amino acid sequences may have a common structural domain and / or common functional activity. For example, amino acid sequences substantially identical to LOX contain a common structural domain with at least about 60% or 65% identity, probably 75% identity, more likely 85%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98% or 99% identity with LOX. For example, amino acid sequences containing a common structural domain with at least about 60% or 65% identity, probably 75% identity, more likely 85%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, or 99% identity with LOXL2 are designated sufficiently or substantially identical. In the context of nucleotide sequences, the term " substantially identical " is used herein to refer to a first nucleic acid sequence which contains a sufficient or a minimum number of nucleotides that are identical to the nucleotides aligned in a second nucleic acid sequence, so that the first and second nucleotide sequences encode a polypeptide having common functional activity or encode a common structural polypeptide domain or a common functional polypeptide activity. For example, nucleotide sequences with at least about 60% or 65% identity, probably 75% identity, more likely 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity provided herein are designated substantially identical. II. Lysyl Oxidase (LOX) and Lisil Oxidase (LOXL) Proteins

Typically, solid tumors contain areas of low oxygen tension (hypoxia). Hypoxic cells pose a major problem in the treatment of cancer because these cells are highly aggressive, metastatic and therapeutically resistant. The underlying mechanisms that contribute to these characteristics are poorly understood. Metastasis poses a particular problem in breast cancer because there is no effective treatment for most of the patients with detectable metastatic breast cancer (Steeg, PS Br. Can. Res. 2 (6): 396-9 (2000)). . The extracellular matrix (ECM) may have a curing influence on tumor cells (Chang and Werb, Trends Cell Biol. 11: S37-43 (2001); and Radisky et al., Semin. )). Hypoxia-exposed mice exhibit tissue-specific increases in the activity of lysyl oxidase (LOX), an amine oxidase that plays a key role in the formation and maintenance of ECM (Brody et al., Am. Rev. Respir Dis. 120: 1289-95 (2001)). A recent microarray study confirmed LOX as a hypoxia induced gene in a variety of cell lines (Denko, NC, Oncogene 22: 5907-14 (2003)). However, a biological role of LOX was not identified under hypoxic conditions. LOX initiates covalent cross-linking of collagens and elastin in ECM, increasing the deposition of the insoluble matrix and tensile strength (Kagan and Li, J. Cell Biochem 88: 660-72 (2003)). LOX expression is essential for normal connective tissue healing and function, and knockout mice die shortly after delivery due to cardiovascular instability (Homstra et al., J. Biol. Chem. 278: 14387-93 (2003)). .

LOW LOX activity is associated with diseases such as Ehler-Danlos syndrome (Pinnell, SR J. Invest Dermatol 79 (Supp 1): 90S-92S (1982) Royce et al., Biochem J. 192: 579-86 (1980), and Khakoo et al., Gen. Genet 51: 109-14 (1997)). Increased LOX activity contributes to fibrotic and tissue remodeling diseases, such as hepatic cirrhosis (Kagan, HM, Pathol Res, Pract 190: 910-0 (1994), Chanki et al., Br. J. Dermatol, 133: 710-5 (1995), and Ooshima and Midorikawa, Jpn Cir., J. 41: 1337-40 (1977)).

High LOX expression correlates with increased preparation in renal cell cancer (Stassar et al., Br. J. Cancer, 85: 1372-82 (2001)) and increased expression of LOX is observed in highly breast cancer cell lines (Kirschmann et al., Breast Cancer Res. Treat, 55: 127-36 (1999)) and Kirschmann et al., Cancer Res. 62: 4478-83 (2002)). In contrast, LOX acts as a tumor suppressor in non-tumorigenic reversers of ras-transformed fibroblasts (Smith-Mungo and Kagan, Matrix Biol., 16: 387-98 (1998)). The loss of LOX is associated with tumorgenesis in several types of cancer, such as gastric, colon and prostate cancer (Ren et al., Cancer Res. 58: 1285-90 (1998); Cxiszar et al., Int. J. Cancer 97: 636-42 (2002), and Kaneda et al., Cancer Res. 64: 6410-5 (2004)). It would thus appear that tumor suppressor role of LOX depends on the cell type and state of transformation. It has recently been shown that the propeptide domain (and not the active enzyme) is responsible for tumor suppressor activities. In breast cancer, increased LOX expression is associated with the initial stromal reaction (Decitre et al., Laboratory 78: 143-51 (1998)) and the treatment with antisense LOX in this type of cancer cell prevents invasion in vitro (Kirschmann et al., Cancer Res. 62: 4478-83 (2002)). The LOXL2 amino acid sequence shares a broad sequence homology with the conserved copper binding and catalytic domains of both LOX and LOXL. These conserved domains are encoded by five consecutive exons within the LOX, LOXL and L0XL2 genes which also maintain preservation of the exon-intron structure. The conservation of the nucleotide and amino acid sequence deduced within the carboxy-terminal end of L0XL2, LOX and LOXL include the copper binding domain (WEWHSCHQHYH) in LOX and LOXL and WIWHDCHRHYH in L0XL2 with the four histidines which provide the nitrogen ligands for the copper coordination complex specific for lysyl oxidase proteins (Krebs and Krawetz, Biochim. Biophys, Acta 1202: 7-12 (1993)). The active site in LOX (DIDCQWWIDITDVXPGNY) and LOXL2 (DIDC-QWVDITDVPPPGDY) contains in each a Tyr (Y) residue at the COOH-terminal end, which participates together with a Lys residue in the formation of the quinone cofactor that is present in these proteins. Ten cysteines characteristic of LOX and LOXL are likewise conserved in LOXL2 (Kagan et al., (1994) in Molecular Biology and Pathology of Elastic Tissue (Mecham, RP and Roberts, L., eds.), Ciba Foundation Symposium Series , Wiley, Chichester, UK). A growth factor and cytokine receptor domain present in the LOX and LOXL proteins was also identified within the amino acid sequence derived from LOXL2. Four replicates of the receptor-rich cysteine receptor domain are also present in LOXL2 (Saito et al., J. Biol. Chem 272: 8157-8160 (1997), Resnick et al., Trends Biochem Sci 19: 5-8 (1994) )).

Three major transcription termination sites were noted within the 3'-UTR domains of LOXL2 cDNA. The first termination site has 690 bp 3 'termination codon, the second site has 740 bp and the final transcription termination site is 900 bp 3' from the termination codon. These mRNAs all have 3'-UTRs that differ slightly in size. Most of the exon-intron boundaries of the LOXL2 gene show the consensus sequence (C / T) AG-exon-GT (A / G). The sizes of the 11 exons of the L0XL2 gene range from 112 to 940 bp. Although the LOXL2 gene has 11 exons, five consecutive exons (exons 6-10), which encode copper binding and catalytic domains, exhibit 84% sequence similarity and the exon sizes are very similar to the corresponding exons of the LOX and LOXL. All other exons in the LOXL2 gene are divergent in both sequence and size. LOXL2 was identified in all tissues with the exception of blood leukocytes. LOXL2 mRNA was detected in the heart, liver and pancreas; the expression is significantly higher in the placenta, prostate, uterus and pancreas (ratios between 2 and 3) compared to the lower expression in the brain, lung, skeletal muscle, thymus and kidney (ratios below 0.5). (Jourdan-Le Saux, et al., J. Biol. Chem., 274 (18): 12939-12944 (1999)). The expression of LOX and different LOXL proteins varies in different diseases. This may be due to a number of reasons, such as the difference in tissue distribution, processing, domains, regulation of activity, as well as other differences between proteins. For example, LOX and LOXL are implicated in fibrotic diseases, such as both LOX and LOXL are highly expressed in myofibroblasts around fibrotic areas (Kagen, Pathol., Res. Pract, 190: 910-919 (1994), Murawaki et al. , Hepatology 14: 1167-1173 (1991), Siegel et al., Proc Natl Acad Sci USA 75: 2945-2949 (1978), Jourdan Le-Saux et al., Biochem.

Biophys. Comm. 199: 587-592 (1994); Kim et al. , J. Cell

Biochem. 72: 181-188 (1999)). LOX and various LOXLs are also implicated in a number of cancers. For example, LOXL and LOXL4 have been shown to be epigenetically silenced and can inhibit the ras / extracellular signal regulated kinase signaling pathway in human bladder cancer (Wu et al., Cancer Res. 67: 4123-4129 (2007)). .

Others have shown selective upregulation and amplification of the LOXL4 gene in squamous cell carcinoma of the head and neck (Gorough et al., J. Pathol., 212: 74-82 (2007)). LOX and LOXL2 were also implicated in a number of tumors, such as colon and esophageal cancers (Csiszar, Prog.Nucl Acid Res. 70: 1-32 (2001)). In breast cancer, members of the LOX and LOXL family were cancer linked (Kirschmann et al., Cancer Res. 62: 448-4483 (2002)).

Lysyl oxidase catalyzes the oxidative deamination of peptidyl lysine and hydroxylysine residues on collagens, and peptidyl lysine residues on elastin. The resulting peptidyl aldehydes condense spontaneously and undergo oxidation reactions to form the covalent crosslinks derived from lysine necessary for the normal structural integrity of the extracellular matrix. In the reaction of lysyl oxidase with its substrates, hydrogen peroxide (H2O2) and ammonia are released in stoichiometric amounts with the peptidyl aldehyde product. See, for example, Kagan et al., J. Cell. Biochem. 88: 660-72 (2003).

Lysyl oxidase is secreted into the extracellular environment where it is then processed by proteolytic cleavage to a 30 kDa functional enzyme and an 18 kDa propeptide. 30 kDa lysyl oxidase is enzymatically active, whereas the 50 kDa proenzyme is not. Pro-collagen C-proteinases process pro-lysyl oxidase into their active form and are products of the Bmpl, Till and T112 genes. The localization of the enzyme is primarily extracellular, although processed lysyl oxidase is also located intracellularly and nuclearly. The sequence coding for the propeptide is moderately (60-70%) conserved between the LOX and LOXL proteins, whereas the sequence coding for the 30 kDa C-terminal region of the proenzyme in which the active site is highly conserved (approximately 95%). See Kagan et al. , J. Cell Biochem. 59: 329-38 (1995). LOX is induced by a number of growth factors and steroids, such as TGF-β, TNF-α and interferon (Csiszar, Prog.Nucl.Acid Res. 70: 1-32 (2001)).

It is known that there are five different lysyl oxidases in both humans and mice, LOX and four related LOX or LOX type proteins (LOXL, LOXL2, LOXL3, LOXL4). LOX and LOX proteins are collectively referred to as " LOX / LOXL " for the purposes of this disclosure. The five forms of lysyl oxidases reside on five different chromosomes. These family members show some overlap in structure and function, but they seem to have distinct functions as well. For example, although the main activity of LOX is the oxidation of specific lysine residues in the collagen and elastin outside the cell, it can also act intracellularly, where it can regulate gene expression. In addition, LOX induces chemotaxis of monocytes, fibroblasts, and smooth muscle cells. In addition, a deletion of LOX in knockout mice appears to be lethal at calving (Hornstra et al., J. Biol. Chem. 278: 14387-14393 (2003)), whereas LOXL deficiency does not cause severe developmental phenotype (Bronson et al., Neurosci Lett. 390: 118-122 (2005)). The main activity of LOX is the oxidation of specific lysine residues in collagen and elastin outside the cell, however, it may also act intracellularly, where it can regulate gene expression (Li et al., Proc. Natl. Acad Sci USA 94 In addition, LOX induces chemotaxis of monocytes, fibroblasts, and smooth muscle cells (Lazarus et al., J. Biol. Chem., 27: 36341-36349 (2000)). Matrix Biol., 14: 727-731 (1995) Nelson et al., Proc. Soc. Exp. Biol. Med. 188: 346-352 (1988)). LOX itself is induced by a number of growth factors and steroids, such as TGF-β, TNF-α and interferon (Csiszar, Prog.Nucl.Acid Res. 70: 1-32 (2001)). Recent studies have attributed other roles to LOX in various biological functions, such as developmental regulation, tumor suppression, cell motility and cellular senescence. The diverse role of LOX, and its recently discovered LOX (LOXL) amino acid family, may play important roles with its intracellular and extracellular locations.

As used herein, " lysyl oxidase " refers to an enzyme that catalyzes the following reaction: peptidyl-L-lysyl-peptide + 02 + H20 - > peptidyl-alisyl-peptide + NH 3 + H 2 O 2. Other synonyms for lysyl oxidase (EC 1.4.3.13) include protein-lysine 6-oxidase and protein-L-lysine: oxygen 6-oxidoreductase (desamination). See, for example, Harris et al. , Biochim. Biophys. Acta 341: 332-44 (1974); Rayton et al. , J. Biol. Chem. 254: 621-26 (1979);

Stassen, Biophys. Acta 438: 49-60 (1976). A quinoprotein containing copper with a tyrosylquinone lysyl adduct in its active site, LOX catalyzes the oxidation of peptidyl lysine to result in the formation of peptidyl alpha-aminoadipic-delta-semialdehyde. Once formed, this semialdehyde may spontaneously condense with neighboring aldehydes or with other lysyl groups to form intra and inter-chain cross-links. See, for example, Rucker et al., Am. J. Clin. Nutr. 67: 996S-1002S (1998). The term " LOX " refers to an enzyme having an amino acid sequence substantially identical to a polypeptide expressed or translated from one of the following sequences: EMBL / N. Genbank accession 0s: M94054 (SEQ ID NO: 10); AAA5 952 5.1 (SEQ ID NO: 11) - mRNA; S45875 (SEQ ID NO:

12); AAB2 354 9.1 (SEQ ID NO: 13) -mRNA; S78694 (SEQ ID NO: 14); AAB21243.1 (SEQ ID NO: 15) -mRNA; AF039291 (SEQ ID NO: 16); AAD0 2130.1 (SEQ ID NO: 17) - mRNA; BC074820 (SEQ ID NO: 18); AAH74820.1 (SEQ ID NO: 19) -mRNA; BC074872 (SEQ ID NO: 20); M84150 (SEQ ID NO: 22); AAA59541.1 (SEQ ID NO: 23) - Genomic DNA. One embodiment

of LOX is human preproprotein lysyl oxidase (hLOX) with an amino acid sequence (SEQ ID NO: 7), a hLOX secreted after cleavage of the signal peptide, such as SEQ. ID No.8 or a mature hLOX after proteolytic processing, such as SEQ. ID No. 9. LOX has highly conserved protein domains, conserved in various species including human, mouse, rat, chicken, fish and Drosophila. The human LOX family has a highly conserved C-terminal region containing the 205 amino acids of the LOX catalytic domain. The conserved region contains the binding to copper (Cu), conserved cytokine receptor (CRL) domain and the cofactor lysyl-tyrosylquinone (LTQ) site. Twelve cysteine residues are also likewise conserved, wherein two of them resides within the prepropeptide region and ten are in the catalytically active form of LOX (Csiszar, Prog., Nucl. Acid Res. 70: 1- 32 (2001)). The conserved region also includes a fibronectin binding domain. The pre-propeptide region of LOX contains the signal peptide and is cleaved, the cleavage site is predicted to be between Cys21-Ala22, to generate a signal sequence peptide and a 48 kDa amino acid propeptide form of LOX, which is still inactive. The propeptide is N-glycosylated during passage through the Golgi apparatus which is secreted into the extracellular environment where the pro-enzyme, or pro-peptide, is cleaved between Glyl68-Aspl69 by a metaloendoprotease, a pro-collagen C- proteinase, which are the products of the Bmpl, Tll11 and T112 genes. BMP I (bone morphogenetic protein I) is a pro-collagen C-proteinase which processes the propeptide to produce a 30 kDa functional enzyme and an 18 kDa propeptide. Sequence coding for the propeptide is moderately (60-70%) conserved, whereas the sequence coding for the 30-kD C-terminal region of the proenzyme in which the active site is located is highly conserved ( about 95%). (Kagan et al., J. Cell Biochem 59: 329-38 (1995)). N-glycosyl units are also subsequently removed. LOX occurs in unprocessed and / or processed (mature) forms. The mature form of LOX is typically active although, in some embodiments, unprocessed LOX is also active.

Particular examples of a LOXL enzyme or protein are described in Moinar et al., Biochim Biophys Acta. 1647: 220-24 (2003); Csiszar, Prog. Nucl. Acid Res. 70: 1-32 (2001); and in WO 01/83702 published November 8, 2001. (Note that in these 3 publications, " LOXL1 " was referred to as " LOXL ", while in the present invention " LOXL " is used to refer to to lysyl oxidase-like proteins in general, not only LOXL1.) These enzymes include LOXL1, encoded by mRNA deposited in GenBank / EMBL BC015090; AAH15090.1; LOXL2, encoded by mRNA deposited in GenBank / EMBL U89942; LOXL3, encoded by mRNA deposited in GenBank / EMBL AF282619; AAK51671.1; and LOXL4, encoded by mRNA deposited in GenBank / EMBL AF 338,441; AAK71934.1. Potential signal peptides similar to those described above for LOX were predicted at the amino terminus of LOXL, LOXL2, LOXL3 and LOXL4. The predicted signal cleavage sites are between Gly25-Gln26 for LOXL, between Ala25-Gln26, for LOXL2 and between Gly25-Ser26 for LOXL3. The consensus for cleavage of BUMP-1 in pro-collagens and pro-LOX is between Ala / Gly-Asp and is often followed by an acidic or charged residue. A potential cleavage site for generating active LOXL is Gly303-Asp304, however, it is then followed by an atypical Pro. LOXL3 also has a potential cleavage site on Gly447-Asp448, which is followed by an Asp, processing at this site can produce an active peptide of similar size to activate LOX. A potential cleavage site of BMP-1 was also identified within LOXL4 at Ala569-Asp570 residues (Kim et al., J. Biol. Chem. 278: 52071-52074 (2003)). LOXL2 can also be cleaved proteolytically in a manner analogous to the other members of the LOXL and secreted family (Akiri et al., Cancer Res. 63: 1657-1666 (2003)).

LOX and LOXL enzymes act through a ping-pong mechanism that can be described by Michaelis-Menten kinetics (see Figure 1).

An example of LOX or LOXL protein includes the enzyme having an amino acid sequence substantially identical to a polypeptide expressed or translated from one of the following sequences: EMBL / accesses to Genbank Nos .: M94054; AAA59525.1-mRNA; S45875; AAB2 354 9.1-mRNA; S78694; AAB21243.1-mRNA; AF039291; AAD02130.1-mRNA; BC074820; AAH74820.1-mRNA; BC074872; AAH74872.1-mRNA; M84150; AAA59541.1-Genomic DNA.

The terms " LOX " and " LOXL " also encompass functional fragments or derivatives that substantially retain the enzymatic activity that catalyzes the deamination of lysyl residues. Typically, a functional fragment or derivative retains at least 50% of 60%, 70%, 80%, 90%, 95%, 99% or 100% of its lysyl oxidation activity. It is also intended that a LOX or LOXL2 protein may include conservative amino acid substitutions that do not substantially alter its activity. Suitable conservative amino acid substitutions are known to those skilled in this art and can be performed, generally, without altering the biological activity of the resulting molecule. Those skilled in this art will recognize that, in general, simple amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity. See, for example, Watson, et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., page 224. Conservative and non-conservative amino acid substitutions have been described above.

One feature not known to be common among LOX and LOXL proteins is the receptor-rich cysteine receptor (SRCR) domains. LOX and LOXL lack SRCR domains, whereas LOXL2, LOXL3 and LOXL4 each have four SRCR domains at the N-terminus. SRCR domains are found in secreted, transmembrane or extracellular matrix proteins. SRCR domains are also known to mediate ligand binding in a number of secreted and receptor proteins (Hoheneste et al., Nat. Struct. Biol. 6: 228-232 (1999); Sasaki et al., EMBO J. 17: 1606-1613 (1998)). Another unique domain of LOXL is the presence of a proline-rich domain (Moinar et al., Biochimica Biophsyica Acta 1647: 220-224 (2003)). The tissue distribution may also differ between LOX and the various LOXLs. LOX is highly expressed in the heart, placenta, testis, lung, kidney and uterus, but marginally in the brain and liver. LOXL1 is expressed in the placenta, kidney, muscle, heart, lung and pancreas, and as with LOX, has much lower expression in the brain and liver (Kim et al., J. Biol. Chem. 270: 7176-7182 ( 1995)). LOXL2 is highly expressed in the uterus, placenta and other organs, but similar to LOX and LOXL, with less expression in the brain and liver (Jourdan Le-Saux et al., J. Biol. Chem. 274: 12939: 12944 (1999 )). LOXL3 is highly expressed in the testes, spleen, and prostate, moderately in the placenta, and not in the liver, whereas LOXL4 is highly expressed in the liver (Huang et al., Matrix Biol.20: 153-157 (2001); Maki and Kivirikko , Biochem J. 355: 381-387 (2001) Jourdan Le-Saux et al., Genomics 74: 211-218 (2001) Asuncion et al., Matrix Biol., 20: 487-491 (2001)). The expression, or implication of LOX and the different LOXL proteins, in diseases can also vary. This may be due to a number of reasons, such as the difference in tissue distribution, processing, domains, regulation of activity, as well as other differences between proteins. For example, LOX and LOXL are implicated in fibrotic diseases, since both LOX and LOXL are highly expressed in the myofibroblasts around fibrotic areas (Kagen, Pathol Res. Pract, 190: 910-919 (1994), Murawaki et al. , Hepatology 14: 1167-1173 (1991), Siegel et al., Proc Natl Acad Sci USA 75: 2945-2949 (1978), Jourdan Le-Saux et al., Biochem Biophys. 199: 587-592 (1994);

Kim et al., J. Cell Biochem. 72: 181-188 (1999)). LOX and the various LOXLs are also implicated in a number of cancers. For example, LOXL and LOXL4 have been shown to be epigenetically silenced and can inhibit the ras / extracellular signal regulated kinase signaling pathway in human bladder cancer (Wu et al., Cancer Res. 67: 4123-4129 (2007) ).

Others have shown selective upregulation and amplification of the LOXL4 gene in squamous cell carcinoma of the head and neck (Gorough et al., J. Pathol., 212: 74-82 (2007)). LOX and LOXL2 were also implicated in a number of tumors, such as colon and esophageal cancers (Csiszar, Prog.Nucl Acid Res. 70: 1-32 (2001)). In breast cancer, LOX and members of the LOXL family were linked to cancer (Kirschmann et al., Cancer Res. 62: 448-4483 (2002)). III. Epithelial-to-mesenchymal transition (EMT) refers to the process by which a cell with a gene expression / epithelial cell phenotype characteristic (i.e., expressing specific proteins, factors and molecules) changes or changes the genes or their expression levels which results in a change in the phenotype of the cell as exhibited by the change or change in the expressed genes.

Epithelial and mesenchymal cells represent distinct lineages, each with a unique gene expression profile that shares specific attributes of each cell type. The conversion of an epithelial cell into a mesenchymal cell requires changes in morphology, cell architecture, adhesion and / or migration capacity. Advanced tumor cells often exhibit a conspicuous down regulation of epithelial markers and a loss of intercellular junctions, resulting in a loss of epithelial polarity and reduced intercellular adhesion. Loss of epithelial features is often accompanied by cell motility and expression of the increased mesenchyme genes. EMT may include loss of contact inhibition, altered growth control and / or enhanced invasiveness (Christiansen and Rajasekaran, Cancer Res., 66 (17): 8319-8326 (2006); and Thiery et al., Curr. Opin Cell Biol., 15: 740-6 (2003)). Molecular and morphological features indicative of EMT correlate with poor histological differentiation, destruction of tissue integrity, and metastasis. EMT provides mechanisms for epithelial cells to overcome the physical limitations imposed on them by intercellular junctions and to adopt a mobile phenotype (Burdsal et al., Development, 118: 829-44 (1993)) and Nieto et al., Mech, Dev., 105 : 27-35 (2001)).

Molecular markers commonly used for EMT include increased expression of N-cadherin and vimentin, nuclear localization of β-catenin and increased production of transcription factors, such as Snaill, Snail2, Twist, EF1 / ZEB1, SIP1 / ZEB2 and / or E47 inhibit E-cadherin production. Phenotypic markers for an EMT include, but are not limited to, an increased capacity for migration and three-dimensional invasion, as well as resistance to apoptosis. These markers were further correlated with EMT induction and an association with carcinogenic phenotypes. The occurrence of TMS during tumor progression allows tumor cells to acquire the ability to infiltrate the surrounding tissue and ultimately metastasize to distant sites. Alterations in gene expression within tumor cells may indicate a progression of epithelial or epithelial type gene expression pattern to a pattern of mesenchymal or mesenchymal type expression. By way of example, identification of E-cadherin loss is correlated with metastatic carcinoma as well as resistance to cancer therapies, such as EGFR inhibitors and IGF-R1 inhibitors. Analysis of many different types of cancer reveals that circulating tumor cells, or those found as micrometastases, evidence the conversion of the mesenchyme based on changes in expression in a set of labels. These markers include, but are not limited to, EGFR, E-cadherin, ErbB3, RAB25, integrin beta 6, cadherin-2, fibroblast growth factor binding protein 1, distal-less homeo1, ZEB1 transcription 8), SIP1 and vimentin. By way of example, an epithelial-like gene expression profile includes increased expression or expression of genes, such as E-cadherin, ErbB3 or EGFR. An epithelial type gene expression profile may include the expression of one or more of these genes, at least two, or at least three such genes.

As with the therapeutically resistant cancers previously described, the expression levels of E-cadherin, ErbB3, RAB25, integrin beta 6, cadherin-2, fibroblast growth factor binding protein 1, distal-less homeo 1, ZEB 1 (transcription factor 8), SIP1, TGF-β, F0XC2, GSK-3B, Smad-3, Pez, Snaill, Snail2 and ILK, and vimentin represent genes which are common to EMT characteristics, as well as to those tumor / epithelial-based cancers that develop resistance to their respective therapies. The present invention also relates in general to a method for treating a cancer patient and particularly a cancer which has undergone EMT. The inventors have discovered that cancers that have experienced EMT or have gone from an epithelial-like gene expression pattern to a mesenchymal-like gene expression pattern are receptive to LOX / LOXL inhibitors.

For evaluation of tumor epithelial cell or mesenchymal biomarker expression, patient samples containing tumor cells, or proteins or nucleic acids produced by these tumor cells, may be used in the methods described, for example, in U.S. Patent Application Publication No. 20070065858. In summary, the level of expression of the biomarker can be assessed by assessing the amount (eg, absolute amount or concentration) of the marker in a sample of the tumor cell, for example, a tumor biopsy obtained from a patient or other patient sample which contains tumor-derived material (e.g., blood, serum, urine or other body fluids or excretions, as described hereinbefore). The cell sample may, of course, be subjected to a variety of well known preparative techniques and post-harvest storage (e.g., nucleic acid and / or protein extraction, fixation, storage, freezing, ultrafiltration, concentration, evaporation, centrifugation, etc.) before evaluating the amount of marker in the sample. Likewise, tumor biopsies may also be subjected to preparative and post-harvest storage techniques, for example, fixation. It is possible to detect expression of biomarker proteins with at least one portion which is displayed on the surface of the tumor cells expressing it. It is a simple matter for the skilled artisan to determine whether a marker protein, or a portion thereof, is exposed on the cell surface. For example, immunological methods may be rutilized to detect such proteins in whole cells, or well-known computerized sequence analysis methods may be used to predict the presence of at least one extracellular domain (i.e. including both secreted proteins and proteins with at least one less a cell surface domain). Expression of a marker protein with at least a portion that is displayed on the surface of a cell expressing it can be detected without necessarily lysing the tumor cell (for example, using a labeled antibody that specifically binds to a cell surface domain of the protein). Expression of biomarkers can be evaluated by any of a wide variety of well known methods for detecting the expression of a transcribed nucleic acid or protein. Non-limiting examples of such methods include, for example, immunological methods for detecting secreted, cell surface, cytoplasmic or nuclear surface proteins, protein purification methods, protein activity or function assays, nucleic acid hybridization methods, transcription methods nucleic acid reverse transcription and nucleic acid amplification methods. Expression of a biomarker may be assessed using an antibody (e.g., a labeled, labeled, fluorophore labeled or labeled labeled antibody with chromophore), an antibody derivative (e.g., an antibody conjugated to a substrate or the protein or ligand of a protein-ligand pair (e.g., biotin-streptavidin) or an antibody fragment (e.g., a single-chain antibody, an isolated antibody hypervariable domain, etc.) which specifically binds to a protein biomarker or fragment thereof, including a biomarker protein which has been subjected to all or a portion of post-translational modifications to which it is normally subjected in the tumor cell (e.g., glycosylation, phosphorylation, methylation, etc.) Expression of a biomarker can also be assessed by preparing mRNA / cDNA (i.e., a transcribed polynucleotide) from cells in a patient sample and hybridizing the mRNA / CDNA with a reference polynucleotide which is a complement of a biomarker nucleic acid, or fragment thereof. The cDNA may optionally be amplified using any of a variety of polymerase chain reaction methods prior to hybridization with the reference polynucleotide. Expression of one or more biomarkers may likewise be detected using quantitative PCR to assess the level of expression of the biomarker (s). Alternatively, any of a number of known methods of detecting mutations or variants (e.g., single nucleotide polymorphisms, deletions, etc.) of a biomarker can be used to detect the occurrence in a patient.

A mixture of transcribed polynucleotides obtained from the sample may be contacted with a substrate having attached thereto a polynucleotide complementary to or homologous with at least one portion (for example, at least 7, 10, 15, 20, 25, 30, 40, 50 , 100, 500 or more nucleotide residues) of a biomarker nucleic acid. If the polynucleotides complementary to, or homologues with, are differentially detectable in the substrate (e.g., detectable using different chromophores or fluorophores or fixed at different selected positions), then the levels of expression of a plurality of biomarkers can be evaluated simultaneously using a single substrate (e.g., a microarray " gene chip " of the polynucleotides attached at selected positions). When a method of evaluating the expression of the biomarker is used which involves hybridization of one nucleic acid with another, hybridization may be performed under stringent hybridization conditions.

When a plurality of biomarkers of the invention are used in the methods of the invention, the level of expression of each biomarker in a patient sample can be compared to the level of normal expression of each of the plurality of biomarkers in non-cancerous samples of the same type, in a single reaction mixture (i.e., using reagents, such as different fluorescent probes, for each biomarker) or in individual reaction mixtures corresponding to one or more of the biomarkers. The level of expression of a normal (i.e., non-cancerous) human tissue biomarker can be evaluated in a variety of ways. This level of normal expression can be assessed by assessing the level of expression of the biomarker in a portion of cells that appear to be non-cancerous and then comparing the level of normal expression with the level of expression in a portion of the tumor cells. As more information becomes available as a result of the routine performance of the methods described herein, population mean values for normal expression of biomarkers may be used. Alternatively, the normal expression level of a biomarker may be determined by evaluating expression of the biomarker in a patient sample obtained from a non-cancer patient from a patient sample obtained from a patient prior to the cancer patient being challenged on the patient , of archived patient samples and the like.

An exemplary method for detecting the presence or absence of a biomarker protein or nucleic acid in a biological sample involves obtaining a biological sample (e.g., tumor associated body fluid) from a test subject and contacting the biological sample with a compost or an agent capable of detecting the polypeptide or nucleic acid (e.g., mRNA, genomic DNA or cDNA). Methods of detection can thus be used to detect mRNA, protein, cDNA or genomic DNA, for example, in a biological sample in vitro as well as in vivo. In vitro techniques for detecting mRNA include, for example, Northern hybridizations and in situ hybridizations. In vitro techniques for detecting a biomarker protein include, but are not limited to, enzyme-linked immunosorbent assays (ELISAs), Western blots, immunoprecipitation and immunofluorescence. In vitro techniques for detecting genomic DNA include, for example, Southern hybridizations. In vivo techniques for detecting mRNA include, for example, polymerase chain reaction (PCR), Northern hybridizations and in situ hybridizations. In addition, in vivo techniques for detecting a biomarker protein include introducing into a subject a labeled antibody directed against the protein or fragment thereof. For example, the antibody may be labeled with a radioactive label whose presence and location in a subject can be detected by conventional tomography techniques.

A general principle of such diagnostic and prognostic assays involves preparing a sample or reaction mixture which may contain a biomarker, and a probe, under appropriate conditions and for a time sufficient to allow the biomarker and the probe to interact and bind, thus, a complex which can be removed and / or detected in the reaction mixture. These assays can be conducted in a variety of ways.

For example, one method of conducting such an assay involves anchoring the biomarker or probe into a solid phase carrier, also referred to as a substrate, and detecting target biomarker / probe complexes anchored in the solid phase at the end of the reaction. In one embodiment of such a method, a sample of a subject, which is to be assayed for the presence and / or concentration of the biomarker, may be anchored in a solid phase carrier or carrier. In another embodiment, the opposite situation is possible, in which the probe may be anchored to a solid phase and a sample of a subject may be allowed to react as a non-anchored component of the assay.

There are several established methods for anchoring test components to a solid phase. These include, without limitation, biomarker or probe molecules that are immobilized by conjugation of biotin and streptavidin. Such biotinylated assay components may be prepared from biotin-NHS (N-hydroxysuccinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.) And immobilized on the wells of 96- wells coated with streptavidin (Pierce Chemical). In certain embodiments, surfaces with immobilized assay components may be prepared in a timely manner and stored. Other solid phase carriers or carriers suitable for such assays include any material capable of binding the class of molecules to which the biomarker or probe belongs. Well known carriers or carriers include, but are not limited to, glass, polystyrene, nylon, polypropylene, nylon, polyethylene, dextran, amylases, natural and modified celluloses, polyacrylamides, gabbros and magnetite. To conduct assays with the aforementioned approaches, the non-immobilized component is added to the solid phase in which the second component is anchored. After the reaction is complete, the non-complexed components may be removed (for example by washing) under conditions such that any complexes formed will remain immobilized on the solid phase. Detection of solid phase anchored / biomarker / probe complexes can be achieved by a number of methods summarized herein. In one embodiment, the probe, when it is the non-anchored assay component, may be labeled for the purpose of detecting and reading the assay, either directly or indirectly, with detectable labels discussed herein and which are well known to one skilled in the art.

It is also possible to directly detect the formation of biomarker / probe complexes without further manipulation or labeling of any component (biomarker or probe), for example using the fluorescence energy transfer technique (i.e., FET, see, for example, Lakowicz et al. al., US Patent No. 5,631,169; Stavrianopoulos, et al., U.S. Patent No. 4,868,103). A fluorophore label on the first donor molecule is selected in such a way that, at the time of excitation with incident light of suitable wavelength, its emitted fluorescent energy will be absorbed by a fluorescent label on a second acceptor molecule which, by its time, it is able to fluoresce because of the energy absorbed. Alternately, the donor protein molecule may simply utilize the natural fluorescent energy of the tryptophan residues. Labels that emit different wavelengths of light are chosen so that the label of the acceptor molecule can be differentiated from that of the donor. Since the efficiency of the energy transfer between the labels is related to the distance separating the molecules, the spatial relationships between the molecules can be evaluated. In a situation where binding between molecules occurs, the fluorescent emission of the acceptor molecule label in the assay should be maximal. A FET binding event can be conveniently measured by conventional fluorometric detection means well known in the art (for example, using a fluorimeter).

In another embodiment, determining the ability of a probe to recognize a biomarker can be achieved without labeling any one component of the assay (probe or biomarker) using a technology, such as Real-Time Biomolecular Interaction Analysis (BIA) (see, for example , Sjolander, S. and Urbaniczky, C., 1991, Anal Chem 63: 2338-2345 and Szabo et al., 1995, Curr. Opin. Struct. Biol., 5: 699-705). As used herein, " BIA " or " surface plasma resonance " is a technology for studying biospecific interactions in real time without labeling any of the interactors (eg, BIAcore). Changes in the bond surface mass (indicator of a bonding event) result in changes in the refractive index of light near the surface (the surface optical resonance phenomenon (SPR)), resulting in a detectable signal that can be used as an indication of real-time reactions between biological molecules.

Alternatively, in another embodiment, diagnostic and prognostic analogue assays with biomarker and probe can be conducted as solutes in a liquid phase. In such an assay, the complexed biomarker and probe are separated from the non-complexed components by any of a number of conventional techniques, including, but not limited to: differential centrifugation, chromatography, electrophoresis, and immunoprecipitation. In differential centrifugation, the biomarker / probe complexes can be separated from the non-complexed components of the assay by a series of centrifugation steps, due to the different sedimentation equilibria of the complexes based on their different sizes and densities (see, for example, Rivas , G., and Minton, AP, 1993, Trends Biochem Sci. 18 (8): 284-7). Conventional chromatographic techniques may also be used to separate complexed molecules from non-complexed molecules. For example, gel filtration chromatography separates molecules based on size and through the use of an appropriate gel filtration resin in a column format; for example, the relatively larger complex may be separated from the relatively smaller non-complexed components. Likewise, the relatively different charge properties of the biomarker / probe complex as compared to the non-complexed components may be exploited to differentiate the complexed components from the non-complexed ones, for example through the use of ion exchange chromatography resins. Such resins and chromatographic techniques are well known to those skilled in the art (see, for example, Heegaard, NH, 1998, J. Mol. Chromatogr B Biomed Sci Appl Oct 10, 1997; 699 (1-2): 499-525). Gel electrophoresis may also be employed to separate complexed components of the assay from unbound components (see, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987-1999 ). In this technique, protein or nucleic acid complexes are separated based on size or charge, for example. To maintain the bonding interaction during the electrophoretic process, gel matrix materials and non-denaturing conditions are typically used in the absence of reducing agent. Suitable conditions for the particular assay and components thereof will be well known to one skilled in the art.

In another embodiment, the level of the mRNA biomarker may be determined both in situ and in vitro formats in a biological sample using methods known in the art. The term " biological sample " it is intended to include tissues, cells, biological fluids and isolates thereof, isolated from a subject, as well as tissues, cells and fluids present within a subject. Many methods of expression detection utilize RNA alone. For in vitro methods, any non-selectable RNA isolation technique against mRNA isolation may be used for the purification of RNA from tumor cells (see, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York 1987-1999). In addition, large numbers of tissue samples can readily be processed using techniques well known to those skilled in the art, such as, for example, the single-step RNA isolation process of

Chomczynski (1989, U.S. Patent No. 4,843,155). The isolated mRNA may be used in hybridization or amplification assays which include, but are not limited to, Southern or Northern analyzes, polymerase chain reaction assays and probes. A diagnostic method for detecting mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) which can hybridize to the mRNA encoded by the gene to be detected. The nucleic acid probe may be, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250, or 500 nucleotides in length and sufficient to hybridize specifically under stringent conditions to an mRNA or genomic DNA encoding a biomarker of the present invention. Other probes suitable for use in the diagnostic assays of the invention are described herein. Hybridization of an mRNA with the probe indicates that the biomarker in question is being expressed.

In one format, the mRNA is immobilized on a solid surface and contacted with a probe, for example, by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probe (s) are immobilized on a solid surface and the mRNA is contacted with the probe (s), for example, in an Affymetrix gene chip array. An skilled artisan can readily adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the biomarkers of the present invention.

An alternative method for determining the level of mRNA biomarker in a sample involves the nucleic acid amplification process, for example, by RT-PCR (the experimental mode presented in Mullis, 1987, U.S. Patent No. 4,683,202), The invention also relates to a method for the preparation of a polypeptide ligase (Barany, 1991, Proc. Natl. Acad Sci USA, 88: 189-193), self-sustained sequence replication (Guatelli et al., 1990, Proc. Natl Acad Sci USA 87: 1874- 1878), transcriptional amplification system (Kwoh et al., 1989, Proc Natl Acad Sci USA 86: 1173-1177), Q-Beta Replicase (Lizardi et al., 1988, Bio / Technology 6: 1197) , roller circle replication (Lizardi et al., U.S. Patent No. 5,854,033) or any other method of nucleic acid amplification, followed by detection of the amplified molecules using techniques well known to those skilled in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present at various low numbers. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5 'or 3' regions of a gene (more or less strands, respectively, or vice versa) and contain a short region between them. In general, the amplification primers are about 10 to 30 nucleotides in length and flank a region of about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers allow the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.

For in situ methods, mRNA need not be isolated from tumor cells prior to detection. In such methods, a cell or tissue sample is prepared and / or processed using known histological methods. The sample is then immobilized on a carrier, typically a glass slide, and then contacted with a probe that can hybridize to the mRNA encoding the biomarker.

As an alternative to making determinations based on the absolute expression level of the biomarker, the determinations may be based on the normalized expression level of the biomarker. Expression levels are normalized by correcting the absolute expression level of a biomarker by comparing its expression with the expression of a gene that is not a biomarker, for example a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes, such as the actin gene or epithelial cell-specific genes. This standardization allows comparison of the level of expression in a sample, for example, in a patient sample, with another sample, for example a non-tumor sample or between samples from different sources.

Alternatively, the level of expression may be provided at a relative level of expression. To determine a relative level of expression of a biomarker (e.g., a mesenchymal biomarker), the level of biomarker expression is determined for 10 or more, 20 or more, 30 or more, 40 or more or 50 or more samples of the isolates of the normal versus cancerous cell prior to the determination of the level of expression for the sample in question. The mean level of expression of each of the genes assayed in the largest number of samples is determined and is used as a standard expression level of value for the biomarker. The level of expression of the biomarker determined for the test sample (absolute expression level) is then divided by the mean expression value obtained for that biomarker. This provides a level of relative expression.

In another embodiment of the present invention, a biomarker protein is detected. One type of agent for detecting a biomarker protein of the invention is an antibody capable of binding to such a protein or a fragment thereof, such as, for example, a detectably labeled antibody. The antibodies may be polyclonal or monoclonal. An intact antibody, or an antigen-binding fragment thereof (e.g., Fab, F (ab ') 2, Fv, scFv, single-stranded polypeptide) may be used. The term " labeled " in relation to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physical binding) a substance detectable to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent which is labeled directly. Examples of indirect labeling include detecting a primary antibody using a fluorescently labeled secondary antibody and final labeling of a biotin DNA probe such that it can be detected with fluorescently labeled streptavidin.

Tumor cell proteins can be isolated using techniques that are well known to those skilled in the art. The methods of isolating proteins employed may, for example, be such as those described in Harlow and Lane (Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).

A variety of formats may be employed to determine whether a sample contains a protein that binds to a given antibody. Examples of such formats include, but are not limited to, enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis and enzyme linked immunosorbent assay (ELISA). A skilled artisan can readily adapt known protein / antibody detection methods for use in determining whether tumor cells express a biomarker of the present invention.

In one format, antibodies, or fragments or antibody derivatives, may be used in methods such as Western blots or immunofluorescence techniques to detect the expressed proteins. In such uses, both antibodies and proteins can be immobilized on a solid support. Solid phase carriers or suitable carriers include any carrier capable of binding to an antigen or to an antibody. Well known carriers or vehicles include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros and magnetite. One skilled in the art will know many other suitable vehicles for attaching the antibody or antigen and will be able to adapt such a carrier for use with the present invention. For example, proteins isolated from tumor cells can be run on polyacrylamide gel electrophoresis and immobilized on a solid phase support, such as nitrocellulose. The carrier can then be washed with suitable buffers followed by treatment with the detectably labeled antibody. The solid phase carrier can then be washed with the buffer a second time to remove unbound antibody. The amount of label attached on the solid support can then be detected by conventional means.

For ELISA assays, the specific binding pairs may be of the immune or nonimmune type. Specific immune binding pairs are exemplified by antigen-antibody systems or hapten / anti-hapten systems. Fluorescein / anti-fluorescein, dinitrophenyl / anti-dinitrophenyl, biotin / anti-biotin, peptide / anti-peptide and the like may be mentioned. The antibody member of the specific binding pair may be produced by standard methods familiar to those skilled in the art. Such methods involve immunizing an animal with the antigen member of the specific binding pair. If the antigen member of the specific binding pair is not immunogenic, for example, a hapten, it may be covalently coupled to a carrier protein to become immunogenic. Non-immune binding pairs include systems in which the two components share a natural affinity for each other, but are not antibodies. Exemplary non-immune pairs are biotin-streptavidin, intrinsic factor-vitamin B 12, folic acid-folate binding protein and the like.

A variety of methods are available for covalently labeling antibodies with members of specific binding pairs. The methods are selected based on the nature of the member of the specific binding pair, the type of binding desired and the tolerance of the antibody to various conjugation chemistries. Biotin can be coupled covalently to antibodies using commercially available active derivatives. Some of these are biotin-N-hydroxysuccinimide which binds to amine groups in proteins; biotin hydrazide which binds to carbohydrate, aldehyde and carboxyl moieties through a carbodiimide linkage; and biotin maleimide and iodoacetyl biotin which binds to sulfhydryl groups. Fluorescein can be coupled to protein amine groups using fluorescein isothiocyanate. Dinitrophenyl groups may be coupled to protein amine groups using 2,4-dinitrobenzene sulfate or 2,4-dinitrofluorobenzene. Other conventional conjugation methods may be employed to attach monoclonal antibodies to a member of a specific binding pair including dialdehyde, carbodiimide coupling, homofunctional crosslinking and heterobifunctional crosslinking. Carbodiimide coupling is an effective method of attaching carboxyl groups to one another to amine groups. Carbodiimide coupling is facilitated using the commercially available reagent 1-ethyl-3- (dimethylaminopropyl) -carbodiimide (EDAC).

Homobifunctional crosslinkers, including the bifunctional imidoesters and N-hydroxysuccinimide bifunctional esters, are commercially available and are employed to join amine groups in one substance to amine groups in another. Heterobifunctional crosslinkers are reagents having different functional groups. The most commonly commercially available heterobifunctional crosslinkers have an amine reactive N-hydroxysuccinimide ester as a functional group and a sulfhydryl group reactive as the second functional group. The most common reactive sulfhydryl groups are maleimides, pyridyl disulfides and active halogens. One of the functional groups may be a photoactive aryl nitride, which when irradiated reacts with a variety of groups. The detectably labeled antibody or detectably labeled member of the specific binding pair is prepared by attaching to a reporter, which may be a radioactive isotope, enzyme, fluorogenic, chemiluminescent or electrochemical material. Two commonly used radioactive isotopes are 1251 and 3H. Conventional radioactive isotope labeling procedures include the methods of chloramine T, lactoperoxidase and Bolton-Hunter for 1251 and reductive methylation for 3H. The term " detectably labeled " refers to a molecule labeled in such a way that it can be readily detected by the intrinsic enzyme activity of the label or by the labeling of another component which can itself be readily detected.

Suitable enzymes for use in this invention include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, luciferases, including firefly and rennet, β-lactamase, urease, green fluorescent protein (GFP), and lysozyme. Enzyme labeling is facilitated using dialdehyde, carbodiimide linkage, homobifunctional crosslinkers and heterobifunctional crosslinkers, as described above, to bind an antibody with a member of a specific binding pair. The labeling method chosen depends on the functional groups available on the enzyme and on the material to be labeled and on the tolerance of both conjugation conditions. The labeling method used in the present invention may be one of, but not limited to, any of the conventional methods employed including those described by

Engvall and Pearlmann, Immunochemistry 8, 871 (1971), Avrameas and Temynck, Immunochemistry 8, 1175 (1975), Ishikawa et al. , J. Immunoassay 4 (3): 209-327 (1983) and Jablonski, Anal. Biochem. 148: 199 (1985). Labeling may be accomplished by indirect methods, such as using spacers or other members of specific binding pairs. An example of this is the detection of a biotinylated antibody with unlabeled streptavidin and biotinylated enzyme, with streptavidin and biotinylated enzyme to be added sequentially or simultaneously. Thus, according to the present invention, the antibody used to detect can be detectably labeled directly with a reporter or indirectly with a first member of a specific binding pair. When the antibody is attached to a first member of a specific binding pair, then the detection is effected by reacting the antibody-first member of a specific binding complex with the second member of the binding pair which is labeled or unlabeled, as mentioned previously.

In addition, the unlabeled detector antibody can be detected by reacting the unlabeled antibody with a labeled antibody specific for the unlabeled antibody. On this occasion, " detectably labeled " as used above, it is interpreted as containing an epitope whereby an antibody specific for the unlabelled antibody can bind. Such an anti-antibody can be labeled directly or indirectly using any of the approaches discussed above. For example, the anti-antibody can be bound to biotin which is detected by reacting with the streptavidin-horseradish peroxidase system discussed above. Thus, in one embodiment, biotin is used. The biotinylated antibody is, in turn, reacted with the streptavidin-strongpoxidase complex. Orthophenylenediamine, 4-chloro-naphthol, tetramethylbenzidine (TMB), ABTS, BTS or ASA may be used for chromogenic detection.

In an immunoassay format for practicing this invention, a forward sandwich assay is used in which the capture reagent has been immobilized using conventional techniques on the surface of a carrier. Suitable carriers used in the assays include synthetic polymer carriers, such as polypropylene, polystyrene, substituted polystyrene, for example, aminated or carboxylated polystyrene, polyacrylamides, polyamides, polyvinyl chloride, glass beads, agarose or nitrocellulose. IV. Anti-LOXL2 Antibodies Antibodies are provided herein which can be used to diagnose angiogenesis and associated diseases, fibrosis and associated diseases, tumors or metastasis. Antibodies are provided herein which inhibit angiogenesis and associated diseases, inhibit fibrosis and associated diseases, and treat tumors or metastasis. Antibodies are provided herein which can be used to monitor the efficacy of treatment regimens and protocols and the like, as described throughout the present application and known in the art. Antibodies and antigen binding fragments useful in such methods are those, for example, that bind specifically to L0XL2. The disclosure also describes cell lines which produce antibodies or functional fragments thereof, methods for producing the cell lines and methods for producing the antibodies or functional fragments thereof. The term " antibody " or " antibody molecule " in the various grammatical forms is used herein as a collective name referring to a population of immunoglobulin molecules and / or immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antibody or paratopotide combining site. Thus, reference to an " antibody " also includes reference to any of the antigen-binding fragments of the antibodies.

As used herein, " immunoreactive " refers to antibodies or fragments thereof that are specific for a sequence of amino acid residues (" binding site " or " epitope "), however if they are cross-reactive with other peptides / proteins, they are non-toxic at the levels for which they are formulated for administration to human use. " Epitope " refers to that portion of an antigen capable of forming a binding interaction with an antibody or antigen-binding fragment thereof. Such binding interaction may be manifested as an intermolecular contact with one or more amino acid residues of a CDR. The antigen binding may involve a CDR3 or a CDR3 pair. An epitope may be a linear peptide sequence (i.e., " continuous ") or may be composed of noncontiguous (ie, " conformational " or " discontinuous ") amino acid sequences. The term " preferably binds " means that the binding agent binds to the binding site with greater affinity than it binds to unrelated amino acid sequences.

As used herein, the term " affinity " refers to the equilibrium constant for the reversible binding of two agents and is expressed as a dissociation constant (Kd). The affinity may be at least 1 fold higher, at least 2 fold higher, at least 3 fold higher, at least 4 fold higher, at least 5 fold higher, at least 6 fold higher, at least 7 fold higher, at least 8 fold at least 9 times higher, at least 10 times, at least 10 times, at least 20 times higher, at least 30 times higher, at least 40 times higher, at least 50 times higher, at least 60 times higher, at least 70 times higher, at least 80-fold, at least 90-fold, at least 100-fold or at least 1000-fold, or more, than the affinity of an antibody for unrelated amino acid sequences. The affinity of an antibody for a target protein may be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM) or about 100 nM to about 1 femtomolar (fM) or more. As used herein, the term " avidity " refers to the resistance of a complex of two or more agents to dissociation upon dilution. The terms " immunoreactive " and " preferably bind " are used interchangeably herein with respect to antibodies and / or antigen-binding fragments. The term " antibody " also includes molecules that have been engineered through the use of molecular biological techniques to include only portions of the native molecule since those molecules have the ability to bind to a particular antigen or amino acid sequences with the required specificity. Such alternative antibody molecules include classically-related portions of the antibody molecules, single chain antibodies and single chain linker molecules.

A " antibody combining site " is that structural portion of an antibody molecule comprising light and heavy chain variable and hypervariable regions that specifically bind to the antigen.

As used herein, the term " CDR " or " complementarity determining region " is intended to mean the non-contiguous antigen-combining sites found within the variable region of both the light and heavy chain polypeptides. These particular regions have been described by Rabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Rabat et al., Department of Health and Human Services U.S., " Sequences of proteins of immunological interest "(1991); by Chothia et al., J. Mol.

Biol. 196: 901-917 (1987); and MacCallum et al., J. Mol. Biol. 262: 732-745 (1996), where the definitions include overlaps or subsets of amino acid residues as compared to each other. However, the application of any of the definitions to refer to a CDR of an antibody or grafted antibodies or variants thereof is intended to be within the scope of the term as defined and used herein. Amino acid residues encompassing the CDRs, as defined by each of the abovementioned references, are presented in Table 1 below as a comparison.

As used herein, the term " structure " when used in reference to an antibody variable region is meant to mean all amino acid residues outside the CDR regions within the variable region of an antibody. A variable region framework is, in general, a discontinuous amino acid sequence of about 100-120 amino acids in length, but is intended to refer only to those amino acids outside the CDRs. As used herein, the term " structure region " is intended to mean each domain of the structure that is separated by the CDRs. " An inhibitor of L0XL2 activity " may be an antibody or an antigen-binding fragment thereof which directly or indirectly inhibits lysyl oxidase activity, including, but not limited to, gene expression, post-translational modification, enzymatic processing or cleavage, binding to a L0XL2 modulator , enzymatic activity of L0XL2 or any other activity described herein.

As used herein, the term " antibody " refers to an isolated or recombinant binding agent comprising the variable region sequences necessary to specifically bind to an antigenic epitope. Therefore, an antibody is any form of antibody or fragment thereof which exhibits the desired biological activity, for example binding to the specific target antigen. Thus, it is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, diabodies, multispecific antibodies (for example, bispecific antibodies), and antibody fragments including but not limited to, single chain binding polypeptides, VH, VL, Fv, scFv, Fab and Fab2, etc., provided they exhibit the desired biological activity. The term " human antibody " therefore refers to antibodies which contain sequences of human origin, except for possible non-human CDR regions, and does not imply that the complete structure of an Ig molecule is present, only that the antibody has minimal immunogenicity in a human. The term " binding " refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic and ionic and / or hydrogen bonding interactions, including interactions such as salt bridges and water bridges. The term " specific binding " is applicable to a situation in which an antibody or antigen-binding fragment thereof does not show any significant binding to other molecules other than its epitope. In one embodiment, an antibody or antigen-binding fragment thereof binds specifically to a human LOX or a human L0XL2 with a Kd dissociation constant equal to or less than about 100 nM, less than about 10 nM, lower to about 1 nM, less than about 0.5 nM, less than about 0.1 nM, less than about 0.01 nM or less than about 0.005 nM measured at a temperature of about 4 ° C, 25 ° C, 37 ° C or 42 ° C.

Conventional methods for preparing antibodies can be used. For example, using an integer peptide or protein lysyl oxidase, polyclonal antisera or monoclonal antibodies can be made using standard methods. A mammal, (e.g., a mouse, hamster or rabbit) may be immunized with an immunogenic form of the peptide which triggers an antibody response in the mammal. Techniques for conferring potentiated immunogenicity in a peptide include conjugation with carriers or other techniques well known in the art. For example, the protein or peptide may be administered in the presence of adjuvant. The progress of immunization can be monitored by detecting the antibody titers in plasma or serum. ELISA procedures or other conventional immunoassays may be used with the immunogen as antigen to assess antibody levels. After immunization, the antisera can be obtained and, if desired, polyclonal antibodies isolated from the sera.

Antibodies can be generated in cell cultures, phage or various animals, including, but not limited to, cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, primates. Therefore, an antibody useful in the present methods is typically a mammalian antibody. Phage techniques may be used to isolate an early antibody or to generate variants with altered specificity or avidity characteristics. Such techniques are routine and well known in the art. In one embodiment, the antibody is produced by recombinant means known in the art. For example, a recombinant antibody can be produced by transfecting a host cell with a vector comprising a DNA sequence encoding the antibody. One or more vectors may be used to transfect the DNA sequence expressing at least one VL region and a VH in the host cell. Exemplary depictions of recombinant antibody generation and production media include Delves, Antibody Production: Essential Techniques (Wiley, 1997); Shephard, et al., Monoclonal antibodies (Oxford University Press, 2000); and Goding, Monoclonal antibodies: Principles and Practice (Academic Press, 1993).

Antibodies may also be made according to the protocol described in Kenney, et al. (" Production of monoclonal antibodies using a secretion capture web report. " Biotechnology 13: 787-790, 1995). In summary, mice are injected subcutaneously (s.c.) with antigen in an adjuvant formulation. For peptide antigens, peptides are conjugated with bovine serum albumin and formulated in Freund's Adjuvant (FA) prior to immunization. For protein antigens, the protein is formulated in alhydrogel-Muramyl Dipeptide (ALD / MDP) adjuvant. Spleen cells and lymph nodes of the mice are isolated and fused with appropriate and cultured cells. A hybridoma library of selected HAT cells is isolated and cloned. Cells are sorted and sera and supernatants are screened for the presence of antibodies. " Antibody fragments " comprise a portion of an intact antibody and may include binding to the antigen or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab') 2, Fv fragments, scFv fragments, diabodies, linear antibodies (Zapata et al., Protein Eng. 8 (10): 1057-1062 (1995)), single chain antibody molecules, single chain binding polypeptides and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen binding fragments, termed " Fab " fragments, each with a single antigen binding site and a " Fc " residual, a designation that reflects the ability to crystallize rapidly. Pepsin treatment produces an F (ab ') 2 fragment which has two antigen-combining sites and is further capable of cross-linking the antigen.

A " single-stranded binding polypeptide " refers to a polypeptide with a heavy chain variable region, a light chain variable region and optionally an Fc region of the immunoglobulin. Such a molecule is a single-stranded variable fragment optionally having an effector function through the presence of the Fc region of the immunoglobulin. Methods of preparing single-stranded polypeptides are known in the art (e.g., U.S. Patent Application No. 2005/0238646). " Fv " refers to an antibody fragment which contains a complete antigen-binding and recognition site. This region consists of a dimer of a light chain variable domain and a heavy chain variable domain in close covalent association. It is in this configuration that the three CDRSs of each variable domain interact to define an antigen binding site on the VH-VL dimer surface. Collectively, the six CDRs confer antigen binding specificity on the antibody. However, even a single variable domain (or half of an Fv comprising only three antigen-specific CDRs) has the ability to recognize and bind the antigen, although at a lower affinity than the complete binding site.

A " Fab " contains an " Fv " and also contains the light chain constant domain and the first constant domain (CHI) of the heavy chain. Fab fragments differ from the Fab 'fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the hinge region of the antibody. Fab'-SH is the designation herein for Fab 'in which the cysteine residue (s) of the constant domains carry a free thiol group. The antibody F (ab ') 2 fragments were originally produced as pairs of Fab' fragments having hinge cysteines therebetween. Other chemical junctions of antibody fragments are also known.

&Quot; light chains " of the antibodies (immunoglobulins) of any vertebrate species can be assigned to one of two distinctly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these can be further subdivided into subclasses (isotypes), for example IgG 1, IgG2, IgG3, IgG4, IgAl and IgA2. The heavy chain (Fc) constant domains corresponding to the different classes of immunoglobulins are designated α, δ, □, e and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

&Quot; light chains " of the antibodies (immunoglobulins) of any vertebrate species can be assigned to one of two distinctly distinct types, called kappa or (" k ") and lambda or (" λ "), based on the amino acid sequences of their constant domains .

Antibody fragments " Single chain Fv " or " sFv " comprise the VH and VL domains of the antibody, wherein these domains are present in a single polypeptide chain. An Fv polypeptide may further include, if necessary, a polypeptide linker between the VH and VL domains, which allows the sFv to form a desired structure for binding to the antigen. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, volume 113, Rosenburg and Moore editors, Springer-Verlag, New York, pages 269-315 (1994). The term " diabodies " refers to small antibody fragments with two antigen-binding sites, the fragments of which contain a heavy chain (VH) variable domain bound to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). Using a linker that is too short to allow pairing between the two domains in the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen binding sites. The diabodies are described in more detail, for example, in EP 404 097; WO 93/11161; and Hollinger et al. , Proc. Nat1. Acad. Know. USA, 90: 6444-6448 (1993).

An antibody can also be a bispecific antibody. Bispecific antibodies are monoclonal, chimeric, human or humanized antibodies having binding specificities by at least two different antigens. In the present case, one of the binding specificities is, for example, LOX or LOXL2, the other is by any other antigen, such as, for example, a cell surface protein or receptor receptor or subunit. In additional embodiments, a bispecific antibody is specific for LOX and LOXL2. Methods for making bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two heavy chain / immunoglobulin light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305: 537-539 (1983)). Because of the random assortment of immunoglobulin light and heavy chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually achieved by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published May 13, 1993 and in Traunecker et al., EMBO J., 10: 3655-3659 (1991).

Variable antibody domains with the desired binding specificities (antibody-antigen-combining sites) may be fused to immunoglobulin constant domain sequences using standard methods known in the art. The fusion is generally with a constant immunoglobulin heavy chain domain containing at least part of the hinge, CH2 and CH3 regions. In one embodiment, the first heavy chain constant region (CHI) containing the site necessary for the light chain binding is present in at least one of the fusions. DNAs encoding the heavy chain fusions of the immunoglobulin and, if desired, the immunoglobulin light chain, may be inserted into separate expression vectors and may be co-transfected into a suitable host organism. For more details on the generation of bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).

According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be manufactured to maximize the percentage of heterodimers that are recovered from the recombinant cell culture. The interface may comprise at least a portion of the CH3 region of a constant domain of the antibody. In this method, one or more small amino acid side chains of the interface of the first antibody molecule are replaced with larger side chains (for example, tyrosine or tryptophan). " Cavities " counterparts of the same size or similar to that of the large side chain (s) are created at the interface of the second antibody molecule by replacing the large side chains of the amino acids with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer on other undesired end products, such as homodimers.

Bispecific antibodies may be prepared as whole-length antibodies or antibody fragments (e.g., Bispecific antibodies F (ab ') 2). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies may be prepared using chemical binding. Brennan et al., Science 229: 81 (1985) describe a procedure in which intact antibodies are proteolytically cleaved to generate F (ab ') 2 fragments. These fragments are reduced in the presence of dithiol sodium arsenite complex agent to stabilize vicinal dithiols and prevent the formation of intermolecular disulfide. The Fab 'fragments generated are then converted to thionitrobenzoate derivatives (TNB). One of the Fab'-TNB derivatives is then reconverted to Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

The Fab 'fragments can be recovered directly from E. coli and chemically linked to form bispecific antibodies. Shalaby et al. , J. Exp. Med. 175: 217-225 (1992) describe the production of a fully humanized bispecific antibody F (ab ') 2 molecule. Each Fab 'fragment was secreted separately from E. coli and subjected to in vitro directed chemical coupling to form the bispecific antibody. The bispecific antibody thus formed was capable of binding to ErbB2 receptor and normal human T cell overexpressing cells, as well as triggering the lytic activity of human cytotoxic lymphocytes against human mammary tumor targets. Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell cultures have also been described. For example, bispecific antibodies were produced using leucine zippers. Kostelny et al., J. Immunol. 148 (5): 1547-1553 (1992). The leucine zipper peptides of the Fos and Jun proteins were linked to the Fab 'portions of two different antibodies by gene fusion. The antibody homodimers were reduced in the hinge region to form monomers and then reoxidized to form the antibody heterodimers. This method can also be used for the production of the antibody homodimers. The " diabody " described by Hollinger et al., Proc. Natl. Acad. Know. USA 90: 6444-6448 (1993) provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy chain variable domain (VH) linked to a light chain variable domain (VL) by a linker that is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thus forming two antigen binding sites. Another strategy for making bispecific antibody fragments by the use of single chain Fv (sFv) dimers has also been reported. See, Gruber et al. , J. Immunol. 152: 5368 (1994).

Antibodies with more than two valences are contemplated. For example, trispecific antibodies may be prepared. Tutt et al., J. Immunol. 147: 60 (1991).

Exemplary bispecific antibodies may bind to two different epitopes in a given LOXL2 polypeptide herein. Alternatively, an anti-LOXL2 arm polypeptide may be combined with an arm that binds to a molecule that triggers a leukocyte such as a T cell receptor molecule (e.g., CD2, CD3, CD28 or B7), or Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD16) in order to focus on cellular defense mechanisms for the cell expressing a particular target polypeptide. Bispecific antibodies may also be used to localize cytotoxic agents to cells expressing a particular target polypeptide. These antibodies have a target binding arm and an arm that binds to a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA or TETA. Another bispecific antibody of interest binds the target polypeptide and binds to tissue factor (TF). The anti-LOXL2 antibody can also be a heteroconjugate antibody. Heteroconjugate antibodies are composed of two covalently linked antibodies. Such antibodies have, for example, been proposed to target cells of the immune system to unwanted cells (U.S. Patent No. 4,676,980) and for treatment of HIV infection (WO 91/00360 and WO 92/200373). Antibodies may be prepared in vitro using methods known in the art of protein synthesis chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or forming a thioether bond. Examples of suitable reagents for this purpose include, but are not limited to, iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.

It may be desirable to modify an anti-LOXL2 antibody with respect to effector function, in order to potentiate, for example, the effectiveness of the antibody in the treatment or prevention of cancer metastasis. For example, cysteine residue (s) may be introduced into the Fc region, thus allowing interchain disulfide bond formation in this region. The thus generated homodimeric antibody may have improved internalization and / or enhanced complement mediated cell death and antibody dependent cellular cytotoxicity (ADCC) capabilities. See Caron et al. , J. Exp. Me., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with enhanced antitumor activity may also be prepared using heterobifunctional crosslinkers as described in Wolff et al. Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody may be engineered to have dual Fc regions and may thus have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).

An " isolated antibody " is one that has been identified and separated and / or recovered from a component of its natural environment. Components of the contaminant from its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include, for example, enzymes, hormones and other proteinaceous or non-proteinaceous solutes. In one embodiment, an antibody will be purified (1) to greater than 80%, 85%, 90%, 95% or 99% by weight antibody as determined by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of N-terminus or internal amino acid sequence through the use of a spinner cup sequenator and / or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver staining. The term " isolated antibody " includes within its scope an in situ antibody within recombinant cells since at least one component of the natural environment of the antibody will not be present. In general, isolation of an antibody or antigen-binding fragment thereof will include at least one purification step.

An antibody may be a humanized antibody or a human antibody. Humanized forms of non-human (e.g., murine) antibodies include, for example, chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, scFv, Fab, Fab ', F (ab') 2, single chain, VH, VL or other antigen-binding subsequences of the antibodies) which contain minimal sequence derived from non-human immunoglobulin. Chimeric antibodies include those in which the variable regions of the light and heavy chains are combined with human constant regions (Fc). Humanized antibodies include human immunoglobulins (receptor antibody) in which residues from a complementarity determining region (CDR) of the receptor are replaced by residues of a CDR from a non-human species (donor antibody) such as mouse, rat or rabbit with the specificity, affinity and capacity. On some occasions residues of the Fv framework of human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or structure sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a sequence human immunoglobulin consensus.

A humanized antibody may also contain at least a portion of an immunoglobulin (Fc) constant region, typically that of a human immunoglobulin (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992)). Methods for humanizing non-human antibodies are well known in the art. In general, a humanized antibody has one or more amino acid residues introduced therein from a source which is non-human. These non-human amino acid residues are often referred to as " imported " or " donors ", which are typically taken from an " imported " variable domain " or " donor ". Humanization can be essentially accomplished following the method of Winter et al. (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988)); Verhoeyen et al. Science, 239: 1534-1536 (1988)), substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such " humanized " include chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are replaced by residues from analogous sites in rodent antibodies.

Human antibodies may also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, J.

Mol Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581 (1991)). The techniques of Cole et al. and Boemer et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal antibodies and Cancer Therapy, Alan R. Liss, 77 (1985) and Boerner et al., J. Immunol., 147 (1): 86 -95 (1991)). Likewise, human antibodies may be made by introducing human immunoglobulin loci into transgenic animals, for example, mice in which endogenous immunoglobulin genes have been partially or completely inactivated. At the time of challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016 and the following scientific publications: Marks et al., Bio / Technology 10,779-783 (1992); Lonberg et al. , Nature 368, 856-859 (1994); Morrison, Nature 368, 81213 (1994); Fishwald et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

Murine monoclonal antibodies have been developed that bind LOX or LOXL2 and block the enzymatic activity thereof. Humanized antibodies and antigen-binding fragments thereof described herein are created by humanization of the VL and VH sequences of anti-LOX and anti-LOXL2 murine monoclonal antibodies.

Humanized immunoglobulins, including humanized antibodies, were constructed by genetic engineering means. Most of the humanized immunoglobulins which have been previously described have comprised a structure which is identical to the structure of a particular human immunoglobulin chain (i.e., an acceptor or receptor) and three CDRs of a non-human (donor) immunoglobulin chain.

As described herein, humanization may also include criteria by which a limited number of amino acids in the structure of a humanized immunoglobulin chain are identified and chosen to be the same as the amino acids at those positions in the donor rather than in the acceptor, so as to increasing the affinity of an antibody comprising the humanized immunoglobulin chain. The present invention is based in part on the model that two contributing causes of loss of affinity in prior media to produce humanized antibodies (using as examples mouse antibodies as the source of CDRs) are: (1) when mouse CDRs are combined with In a human framework, the amino acids in structures close to the CDRs become human rather than mouse. Without being bound by theory, these altered amino acids may slightly distort CDRs (for example, they may create different electrostatic or hydrophobic forces than in donor mouse antibody and distorted CDRs may not make as effective antigenic contacts as CDRs donor antibody); (2) In addition, the amino acids in the original mouse antibody that are close but not part of the CDRs (i.e., still part of the framework) may make contacts with the antigen which contribute to the affinity. These amino acids are lost when the antibody is humanized because, in general, all amino acid structure are made human. To overcome these problems, and to produce humanized antibodies having a very strong affinity for a desired antigen, humanized antibodies and antigen-binding fragments thereof can be constructed using one or more of the following principles.

One principle is that as acceptor, a framework is used of a particular human immunoglobulin that is unusually homologous with the donor immunoglobulin to be humanized or use a consensus framework of many human antibodies is used as an acceptor. For example, comparison of the sequence of a variable region of the mouse heavy (or light) chain against variable heavy (or light) human variable regions in a database (for example, in the Protein Identification Resource of the National Biomedical Research Foundation or the Protein Sequence Database of the National Center for Biotechnology Information (NCBI) shows that the extent of homology with different human regions can vary greatly, for example from about 40% to about 60%, about 70% , about 80% or more. By choosing as the immunoglobulin acceptor one of the human heavy chain variable regions that is most homologous to the donor immunoglobulin heavy chain variable region, fewer amino acids will be altered as they pass from the donor immunoglobulin to the humanized immunoglobulin. By choosing as the immunoglobulin acceptor one of the human light chain variable regions which is most homologous to the variable region of the donor immunoglobulin light chain, fewer amino acids will be altered as they pass from the donor immunoglobulin to the humanized immunoglobulin. In general, using such techniques, there is a reduced possibility of altering an amino acid close to one or more of the CDRs that distort its conformation. In addition, the overall form of a humanized antibody comprising the humanized immunoglobulin chain may appear more closely in the form of the donor antibody, thereby also reducing the possibility of distorting CDRSs.

One may also utilize the light and heavy chains of the same human antibody as acceptor sequences to improve the likelihood that the humanized light and heavy chains will make the contacts favorable to each other. Alternatively, one may also utilize light and heavy chains of different sequences of human antibody germ lines as acceptor sequences; when such combinations are used, one can readily determine whether VH and VL bind an epitope of interest using standard assays (e.g., an ELISA). In one example, the human antibody will be chosen in which the sequences of the heavy and light chain variable regions together are globally more homologous with the sequences of the heavy and light donor variable chain regions. Weight will sometimes be given greater than the heavy chain sequence. Regardless of how the acceptor immunoglobulin is chosen, higher affinity can be achieved in some cases by selecting a small number of amino acids in the humanized immunoglobulin chain structure to be the same as the amino acids at those positions in the donor rather than the acceptor. Affinity maturation methods are known in the art.

Humanized antibodies generally have at least three potential advantages over chimeric or mouse antibodies for use in human therapy. Because the effector portion of an antibody is human, it is believed to interact better with the other parts of the human immune system (e.g., target cell destruction more efficiently by complement dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC )). In addition, the human immune system should not recognize the constant structure or region of the humanized antibody as foreign and therefore the antibody response against such injected antibody should be less than against a totally foreign mouse antibody or a partially foreign chimeric antibody. Finally, mouse antibodies are known to have a half-life in the human circulation that is much shorter than the half-life of human antibodies. Humanized antibodies may presumably have a half-life more similar to naturally occurring human antibodies, allowing the administration of smaller and less frequent doses. Humanization of the antibodies and antigen-binding fragments thereof can be accomplished by a variety of methods known in the art and described herein. Likewise, the production of humanized antibodies can also be achieved by methods known in the art and described herein. Methods for modifications of framework regions are known in the art and are contemplated herein. The selection of one or more relevant amino acid structure positions to be changed depends on a variety of criteria. One criterion for selecting the relevant amino acid structure change may be the relative differences in the amino acid structure residues between the donor and acceptor molecules. The selection of relevant structure positions to be altered using this approach has the advantage of avoiding any subjective bias in the determination of residues or any bias in the contribution of the binding affinity of the CDR for the residue.

Another criterion that can be used to determine the relevant amino acid positions to change may be, for example, selection of structure residues which are known to be important or contribute to the conformation of CDRs. For example, residues of canonical structure are important for conformation and / or CDR structure. The direction of a residue of canonical structure as a relevant position to be altered can be used to identify a more compatible amino acid residue in the context with its associated donor CDR sequence. The frequency of an amino acid residue at a particular structure position is another criterion that may be used to select relevant amino acid structure positions to be altered. For example, comparison of the selected structure with other structure sequences within its subfamily may reveal residues occurring at lower frequencies in a particular position or positions. The positions harboring less abundant residues are likewise applicable for selection as a position to be altered in the structure of the acceptor variable region.

The positions of the relevant amino acids to be altered may also be selected, for example, based on the proximity to a CDR. In certain contexts, FR residues may participate in the conformation and / or binding to the CDR antigen. In addition, this criterion can likewise be used to prioritize relevant positions selected by other criteria described here. Therefore, differentiating between near and distal residues of one or more CDRs represents a way to reduce the number of relevant positions to change.

Other criteria for selecting relevant amino acid structure positions to be altered include, for example, residues that are known to or intended to reside in a three-dimensional space close to the antigen-CDR interface or predicted to modulate the CDR activity. Likewise, structure residues which are known to, or predicted, to form contacts between the heavy chain (VH) and light (VL) variable region interface may be selected. Such structure positions may affect the conformation and / or affinity of a CDR by modulating CDR binding pocket, antigen (epitope) interaction or VH and VL interaction. Therefore, the selection of these amino acid positions to construct a diverse population to classify the binding activity can be used to identify structure changes that replace residues with detrimental effects on CDR conformation or compensate for deleterious effects of residues occurring elsewhere in the structure.

Other structure residues that may be selected for alteration include amino acid positions that are inaccessible to the solvent. Such residues are generally buried in the variable region and are therefore capable of influencing CDR conformation or VH interactions and VL interactions. Accessibility to the solvent can be predicted, for example, from the relative hydrophobicity of the environment created by the amino acid side chains of the polypeptide and / or the known three-dimensional structural data.

Upon selection of relevant amino acid positions in the donor CDRs, as well as any relevant amino acid positions in the desired framework regions to be varied, amino acid changes may be incorporated into some or all of the selected positions in nucleic acids encoding the framework of the variable region acceptor and donor CDRs. The altered CDR framework or sequences may be individually made and tested or may be sequentially or simultaneously combined and tested. The variability at any or all altered positions can range from a few to a plurality of different amino acid residues, including all twenty naturally occurring amino acids or functional equivalents and analogues thereof. In some instances, non-naturally occurring amino acids may also be considered and are known in the art. Selection of the number and location of amino acid positions to be varied is flexible and may depend upon the intended use and desired efficiency for identifying the altered variable region with a desirable activity such as substantially the same binding affinity or higher compared to the donor variable region . In this regard, the greater the number of changes that are incorporated into a population of altered variable regions, the more efficient it is to identify at least one species exhibiting a desirable activity, for example, a binding affinity substantially the same or higher than that of the donor. Alternatively, where the user has empirical or actual data regarding the effect that certain amino acid residues or positions contribute disproportionately to the binding affinity, then it may be desirable to produce a limited population of altered variable regions that focuses on the changes in or around those affinities waste or identified positions.

For example, if CDR-grafted variable regions are desired, a large, diverse population of altered variable regions may include all non-identical framework region positions between the donor and acceptor structure and all positional changes of the single CDR amino acids. Alternatively, a population of intermixture diversity may include subsets, for example, of only the non-identical framework positions close to being incorporated together with all positional changes of single CDR amino acids to, for example, increase the affinity of the humanized antibodies or fragments antigen binding. The diversity of the above populations may further be increased including, for example, additionally all changes in position of the paired CDR amino acids. In contrast, populations that focus on predetermined residues or positions incorporating variant residues to as few as a CDR amino acid structure and / or position can likewise be constructed to classify and identify an antibody variable region changed. As with prior populations, the diversity of such focused populations can be further increased by further expanding the positions selected for change to include other relevant positions in one or both of the framework and CDR regions. There are numerous other combinations ranging from few changes to many changes in one or both of the framework regions and CDRs that may be employed, all of which will result in a population of altered variable regions that can be classified for the identification of at least an altered variable region of CDR grafted with desired activity, e.g., L0X / L0XL2 binding activity. Those skilled in the art will know, or can determine, which residue positions selected in the structure or donor CDRs, or subsets thereof, can be varied to produce a population for classification and identification of an antibody of the invention, given the teachings and guidance provided on here. Codons encoding amino acids are known in the art.

Humanized antibodies and antigen-binding fragments can be made using conventional techniques known in the art. In addition, recombinantly prepared antibodies can be produced, often in large amounts, particularly when using high level expression vectors.

Antibodies can be sequenced using conventional techniques known in the art and the amino acid sequences of the given complementarity determining regions (CDRs). In one aspect, the amino acid sequences of one or more of the CDRs are inserted into a synthetic sequence of, for example, a human antibody framework (or antigen-binding fragment thereof) to create a human antibody that could limit adverse side reactions of treating a human patient with a non-human antibody. The amino acid sequences of one or more of the CDRs may also be inserted into a synthetic sequence of, for example, a binding protein, such as an Avimer to create a construct for administration to a human patient. Such techniques may be modified depending on the species of animal to be treated. For example, for veterinary uses, an antibody, an antigen-binding fragment or binding protein may be synthesized for administration of a primate, a cow, a horse, etc.

In another aspect, using techniques recognized in the art, such as those provided and incorporated herein, nucleotides encoding amino acid sequences of one or more of the CDRs may be inserted, for example, by recombinant techniques at restriction endonuclease sites of an existing polynucleotide which encodes an antibody, an antigen binding fragment or binding protein.

For high-level production, the most widely used mammalian expression system is one which utilizes the gene amplification procedure provided by dihydrofolate reductase deficient Chinese hamster ovary cells (" dhfr "). The system is well known to the skilled craftsman. The system is based on the dihydrofolate reductase gene " dhfr " which encodes the DHFR enzyme, which catalyzes the conversion of dihydrofolate to tetrahydrofolate. To achieve high throughput, dhfr-CHO cells are transfected with an expression vector that contains a functional dhfr gene, along with a gene encoding a desired protein. In this case, the desired protein is heavy chain and / or light chain of the recombinant antibody.

By increasing the amount of the competitive inhibitor DHFR methotrexate (MTX), the recombinant cells develop resistance by amplifying the dhfr gene. In conventional cases, the amplification unit employed is much larger than the size of the dhfr gene, and as a result, the antibody heavy chain is co-amplified.

When large-scale production of the protein, such as the antibody chain, is desired, both the level of expression and the stability of the cells to be employed are taken into account. In long-term culture, recombinant CHO cell populations lose homogeneity relative to their specific antibody productivity during amplification, even though they derive from a single parental clone. The present application provides an isolated polynucleotide (nucleic acid) encoding an antibody or antigen-binding fragment as described herein, vectors containing such polynucleotides and host cells, and expression systems for transcribing and translating such polynucleotides into polypeptides. The present application also provides constructs in the form of plasmids, vectors, transcription or expression cassettes comprising at least one polynucleotide, as above. The present application also provides a recombinant host cell comprising one or more constructs, as above. A nucleic acid encoding any antigen-binding antibody or fragments thereof as described herein as provided, itself forms an aspect of the present application, such as a method of producing the antibody or antigen-binding fragments thereof described herein wherein The method comprises expression of the nucleic acid encoding therefrom. Expression can be conveniently achieved by culturing under appropriate conditions recombinant host cells containing the nucleic acid. After production by expression, an antibody or antigen-binding fragment can be isolated and / or purified using any suitable technique, then used as appropriate.

Antibodies, antigen binding fragments, and specific nucleic acid molecules and vectors described herein may be provided isolated and / or purified, for example, from their natural environment, in substantially pure or homogeneous form or, in the case of the nucleic acid , free or substantially free of nucleic acid origin or genes in addition to the sequence encoding a polypeptide with the necessary function. Nucleic acid may comprise DNA or RNA and may be fully or partially synthetic.

Systems for cloning and expressing a polypeptide in a variety of different host cells are well known. Suitable host cells include cells from bacteria, mammals, yeast systems and baculoviruses. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells and many others. A common bacterial host is E. coli. Expression of antibodies and antibody fragments in prokaryotic cells, such as E. coli, is well established in the art. For a review, see, for example, Pliickthun, A. Bio / Technology 9: 545-551 (1991). Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for producing the antibodies and antigen-binding fragments described herein, see for recent reviews, for example Raff, M.E. (1993) Curr. Opinion Biotech. 4: 573-576; Trill J.J. et al. (1995) Curr. Opinion Biotech 6: 553-560.

Suitable vectors may be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. The vectors may be viral, e.g. phage or phagemid plasmids, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells, and gene expression and protein analysis are described in detail in Short Protocols in Molecular Biology, Second Edition, Ausubel et al. editors, John Wiley & Sons, 1992.

Thus, an additional aspect provides a host cell containing nucleic acid as disclosed herein. Yet another further aspect provides a method which comprises introducing such nucleic acid into a host cell. The introduction may employ any available technique. For eukaryotic cells, suitable techniques may include, for example, calcium phosphate transfection, DEAE Dextran, electroporation, biolistics, liposome-mediated transfection and transduction using retroviruses or other viruses, for example, vaccinia or, for insect cells, baculoviruses. For bacterial cells, suitable techniques may include, for example, transformation with calcium chloride, electroporation and transfection using bacteriophage. The introduction can be followed by causing or allowing expression from the nucleic acid, for example, by culturing host cells under conditions for expression of the gene.

In one embodiment, the nucleic acid is integrated into the genome (e.g., chromosome) of the host cell. Integration can be promoted by including sequences that promote recombination with the genome, according to standard techniques. The present application also provides a method which comprises using a construct, as indicated above, in an expression system to express the antibodies or antigen-binding fragments thereof, as above. The present application also relates to isolated nucleic acids, such as recombinant DNA molecules or cloned genes, or degenerate variants thereof, mutants, analogs or fragments thereof, which encode an antibody or antigen binding sequence which binds to LOX or L0XL2 described herein.

In one aspect, the present application provides a nucleic acid encoding an antibody or antigen-binding fragment thereof which binds to LOX or L0XL2, as described herein.

In a further embodiment, the entire DNA sequence of the recombinant DNA molecule or cloned gene of an antigen-binding antibody or fragment described herein may be operably linked to an expression control sequence that may be introduced into an appropriate host. The request accordingly extends to unicellular hosts transformed with the cloned gene or recombinant DNA molecule which comprises a DNA sequence encoding VH and / or VL, or portions thereof, of the antibody.

Another feature is the expression of the DNA sequences disclosed herein. As is well known in the art, DNA sequences can be expressed by operably linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate unicellular host.

Such operative linkage of a DNA sequence to an expression control sequence obviously includes, if not already part of the DNA sequence, the provision of an initiation codon, ATG, in the correct reading frame upstream of the sequence of DNA.

Polynucleotides and vectors can be provided in an isolated and / or purified form (for example, free or substantially free of polynucleotides of origin other than the polynucleotide encoding a polypeptide with the necessary function). As used herein, " substantially pure " and " substantially free " refer to a solution or suspension containing less than, for example, 20% or less of extrinsic material, 10% or less of extrinsic material, 5% or less of extrinsic material, 4% or less of extrinsic material, 3% or less of extrinsic material, 2% or less of extrinsic material or 1% or less of extrinsic material.

A wide variety of host / expression vector combinations may be employed in the expression of DNA sequences of this invention. Useful vectors include SV40 derivatives and known bacterial plasmids, for example, E. coli col El, Perl, Pbr322, Pmb9 plasmids and derivatives thereof, plasmids such as RP4; Phage DNAs, for example, the numerous λ phage derivatives, for example, NM989 and other phage DNA, for example, M13 and filamentous single-stranded phage DNA; yeast plasmids, such as plasmid 2u or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and others.

Also provided herein is a recombinant host cell comprising one or more polynucleotide constructs. A polynucleotide encoding an antibody or antigen-binding fragment, as provided herein, forms an aspect of the present application, such as a method of producing the antibody or the antigen-binding fragment which method comprises expression from the polynucleotide. Expression can be achieved, for example, by culturing under appropriate conditions recombinant host cells containing the polynucleotide. An antibody or antigen-binding fragment may then be isolated and / or purified using any suitable technique and used as appropriate.

Any of a wide variety of expression control sequences - sequences that control the expression of a DNA sequence operably linked thereto - may be used in these vectors to express the DNA sequences. Such useful expression control sequences include, for example, the initial or late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the LTR system, the regions the β-phage promoter and main promoter, the fd coat protein control regions, the 3-phosphoglycerate kinase promoter or other glycolytic enzymes, acid phosphatase promoters (e.g., Pho5), promoters of yeast and other sequences known to control the expression of prokaryotic or eukaryotic cell genes or their viruses and various combinations thereof.

Systems for cloning and expressing a polypeptide in a variety of different host cells are well known. Suitable host cells include cells from bacteria, mammals, yeast systems and baculoviruses. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells and many others. A common bacterial host may be, for example, E. coli.

Expression of antibodies or antigen-binding fragments in prokaryotic cells, such as E. coli, is well established in the art. For a review, see, for example, Pluckthun, A. Bio / Technology 9: 545-551 (1991). Expression in eukaryotic cells in culture is also available to those skilled in the art (Raff, ME (1993) Curr. Opinion Biotech 4: 573-576; Trill JJ et al. (1995) Curr. Opinion Biotech 6: 553-560) .

A wide variety of unicellular host cells are also useful in the expression of DNA sequences. Such hosts include well known eukaryote and prokaryote hosts such as E. coli strains, Pseudomonas, Bacillus, Streptomyces, fungi, such as yeast and animal cells, such as CHO, YB / 20, NSO, SP2 / 0, RL cells. 1, BW and LM, African green monkey kidney cells (for example, COS 1, COS 7, BSC 1, BSC 40 and BMT 10), insect cells (e.g., Sf 9), and human cells and plant cells in tissue culture .

It will be understood that not all vectors, expression control sequences and hosts will function equally well for expressing the DNA sequences. Not all hosts will work equally well with the same expression system. However, one skilled in the art will be able to select the vectors, expression control sequences and appropriate hosts without undue experimentation to achieve the desired expression without departing from the scope of this application. For example, when selecting a vector, the host must be considered because the vector must work on it. The number of copies of the vector, the ability to control that number of copies and the expression of any other proteins encoded by the vector, such as antibiotic labels, will also be considered. One of ordinary skill in the art can select vectors, expression control sequences and hosts suitable for achieving the desired expression without departing from the scope of this application. For example, when selecting a vector, the host is considered because the vector works on it. The number of copies of the vector, the ability to control that number of copies and the expression of any other proteins encoded by the vector, such as antibiotic labels, can also be considered. The present application also provides constructs in the form of plasmids, vectors, transcription cassettes or expression, as described elsewhere herein, which comprise at least one polynucleotide, as above. Suitable vectors may be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, selectable markers, and other sequences as appropriate. Vectors may be plasmids, viral, for example, phage, phagemid, etc., as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells, and gene expression and protein analysis are described in detail in Short Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992.

When selecting an expression control sequence, a variety of factors will normally be considered. These include, for example, the relative strength of the system, its controllability and compatibility with the particular DNA sequence or gene to be expressed, particularly with respect to potential secondary structures. Suitable unicellular hosts will be selected by consideration of, for example, their compatibility with the chosen vector, their secretion characteristics, their ability to correctly fold proteins and their fermentation requirements, as well as the host toxicity of the product encoded by the sequences of DNA to be expressed and the ease of purification of the expression products.

An additional aspect provides a host cell containing one or more polynucleotides as disclosed herein. A further aspect provides a method of introducing such one or more polynucleotides into a host cell, any available technique. For eukaryotic cells, suitable techniques may include, for example, calcium phosphate transfection, DEAE Dextran, electroporation, liposome-mediated transfection and transduction using retroviruses or other viruses (for example vaccinia) or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include, for example, transformation with calcium chloride, electroporation and transfection using bacteriophages. The introduction can be followed by causing or allowing the expression of one or more polynucleotides, for example, by culturing host cells under conditions for expressing one or more polypeptides of one or more polynucleotides. Inducible systems can be used and expression induced by the addition of an activator.

In one embodiment, the polynucleotides may be integrated into the genome (e.g., chromosome) of the host cell. Integration can be promoted by including sequences that promote recombination with the genome, according to standard techniques. In another embodiment, the nucleic acid is maintained in an episomal vector in the host cell. Methods are provided herein which include using a construct, as indicated above, in an expression system to express a specific polypeptide.

Considering these and other factors, one skilled in the art will be able to construct a variety of vector / host control / vector control sequences that will express the DNA sequences in fermentation or in large scale animal cultures.

A polynucleotide encoding an antibody, antigen-binding fragment or a binding protein may be prepared recombinantly / synthetically in addition to, or instead of, cloned. The polynucleotide may be designed with the appropriate codons for the antibody, antigen-binding fragment or a binding protein. In general, one will select preferred codons for a desired host if the sequence is used for expression. The complete polynucleotide can be assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, for example, Edge, Nature, 292: 756 (1981); Nambair et al. , Science, 223: 1299 (1984); Jay et al. , J. Biol. Chem., 259: 6311 (1984).

A general method for site-specific incorporation into unnatural amino acids into proteins is described in Christopher J. Noren, Spencer J. Anthony-Cahill, Michael C. Griffith, Peter G. Schultz, Science, 244: 182-188 (April 1989 ). This method can be used to create analogs with unnatural amino acids.

As mentioned above, a DNA sequence encoding an antibody or antigen-binding fragment thereof may be prepared synthetically rather than being cloned. The DNA sequence may be designed with the appropriate codons for the amino acid sequence of the antibody or antigen-binding fragment. In general, one will select preferred codons for the intended host if the sequence is used for expression. The complete sequence is assembled from overlapping oligonucleotides by standard methods and assembled into a complete coding sequence. See, for example, Edge, Nature, 292: 756 (1981); Nambair et al. , Science, 223: 1299 (1984); Jay et al., J. Biol. Chem., 259: 6311 (1984). The term " adjuvant " refers to a compound or mixture that potentiates the immune response, particularly an antigen. An adjuvant may serve as a tissue deposit that slowly releases the antigen and also as an activator of the lymphoid system that potentiates the immune response (Hood et al., Immunology, Second Edition, 1984, Benjamin / Cummings: Menlo Park , California, page 384). Often, a primary challenge with only one antigen, in the absence of an adjuvant, will fail to trigger a humoral or cellular immune response. Adjuvants previously known and used include, but are not limited to, Freund's complete adjuvant (CFA), Freund's incomplete adjuvant (IFA), saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, polyols polyanions, peptides, oily or hydrocarbon emulsions, keyhole limpet hemocyanins, dinitrophenol and potentially useful human adjuvant, such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum. Additives of mineral salts include, but are not limited to: aluminum hydroxide, aluminum phosphate, calcium phosphate, zinc hydroxide and calcium hydroxide. Preferably, an adjuvant composition further comprises a fat emulsion lipid comprising about 10% (by weight) vegetable oil and about 1-2% (by weight) phospholipids. Preferably, an adjuvant composition optionally further comprises an emulsion form with oily particles dispersed in a continuous aqueous phase with an emulsion forming polyol in an amount from about 0.2% (by weight) to about 49% (by weight), optionally a metabolizable oil in an emulsion-forming amount of up to 15% (by weight) and optionally a glycol ether-based surfactant in an emulsion stabilizing amount of up to about 5% (by weight).

Antibodies may also be matured for affinity using known selection and / or mutagenesis methods as described above. Affinity matured antibodies may have an affinity that is twice, five times, 10-fold, 20-fold, 30-fold or greater than the starting antibody (generally murine, rabbit, chicken, humanized or human) from which the matured antibody is prepared. Apparent affinities may be determined by methods, such as an enzyme linked immunosorbent assay (ELISA) or any other technique familiar to one skilled in the art. The avides can be determined by methods, such as a Scatchard analysis or any other technique familiar to one skilled in the art. Another technique for measuring apparent affinity of family binding to one skilled in the art is a surface plasma resonance technique (analyzed in a BIACORE 2000 system) (Liljeblad, et al., Glyco, 2000, 17: 323-329) . Standard measurements and traditional binding assays are described by Heeley, R.P., Endocr. Res. 2002, 28: 217-229.

In yet another embodiment, an antibody specifically and selectively binds LOXL2 with a higher binding affinity (e.g., at least about 5-fold, at least about 10-fold, at least about 50-fold, at least about 100 times at least about 500-fold or at least about 1000-fold higher) than the binding affinity for at least one of: human LOX, active or mature human LOX, the secreted form of LOX or other lysyl oxidase-like proteins (LOL) or related lysyl oxidase (e.g. LOXL1, LOXL3 and LOXL4) in unprocessed, mature, active and / or segregated products. In one embodiment, the antibody binds specifically and selectively to LOXL2 in unprocessed and / or processed (mature) forms. The mature form of LOXL2 is typically active although, in some embodiments, unprocessed LOXL2 is also active.

An antibody may bind to both human LOX or human L0XL2, with a higher binding affinity (for example, at least about 5-fold, at least about 10-fold, at least about 50-fold, at least about 100 times, at least about 500-fold or at least about 1000-fold higher) than the binding affinity to at least one of: other lysyl oxidase or lysyl oxidase-related proteins (e.g., LOXL1, LOXL3 and LOXL4).

Antibodies that bind to enzymes may be competitive inhibitors, competitive inhibitors or noncompetitive inhibitors. In relation to competitive inhibition, an inhibitor normally bears structural similarity to the substrate. Inhibition will be evident at low substrate concentrations, but may be overcome at elevated substrate concentrations. With respect to thecompetitive inhibition, an inhibitor binds at the site which becomes available after the substrate is bound in the active site. Inhibition will be more evident at elevated substrate concentrations. In relation to the noncompetitive inhibition, an inhibitor binds at a site far from the substrate binding site. The relative inhibition will generally be the same for all substrate concentrations. The mechanism of action of antibodies that act as competitive inhibitors, competitive inhibitors and noncompetitive inhibitors is illustrated in Figure 4. Antibodies described herein may be competitive inhibitors, competitive inhibitors or noncompetitive inhibitors. In one aspect, antibodies described herein may be non-competitive inhibitors.

Antibodies described herein are noncompetitive inhibitors; that is, antibodies block the enzymatic activity of LOXL2 regardless of whether or not the enzymes are bound to the substrate (collagen). Binding of an antibody to L0XL2 may either (1) reduce or inhibit the uptake or internalization of L0XL2 (for example, through the beta 1 integrin or other cellular receptors or proteins) and / or (2) reduce or inhibit the enzymatic activity of L0XL2 . Such antibody is believed to reduce EMT and is thus useful for the applications disclosed herein. An antibody described herein may bind to the proteolytic cleavage site of L0XL2, thereby effectively blocking (inhibiting) the processing of L0XL2 to reduce the level of active LOX or L0XL2. Such inhibition may occur through direct binding to L0XL2 or through indirect interference including steric hindrance, L0XL2 enzymatic alteration, inhibition of transcription or translation, destabilization of mRNA transcripts, weakened export, processing or localization of L0XL2 and the like. Binding of L0XL2 to other proteins, such as cell receptors (e.g., integrin receptor 1 receptor), BTK (Burton's agammaglobulinemia tyrosine kinase) or other integrins is also performed using the previously mentioned assay, wherein instead of proteins ECM, cell receptors (e.g., integrin receptor 1 receptor), BTK (Burton's agammaglobulinemia tyrosine kinase) or other integrins are used.

Those anti-L0XL2 antibodies which inhibit L0XL2 binding to ECM proteins, cellular receptors and integrins, are selected as candidates for further development. In one embodiment, the anti-L0XL2 antibodies that inhibit L0XL2 binding to ECM proteins, cellular receptors and integrins are non-competitive inhibitors.

In one embodiment, an antibody described herein specifically binds to the LOX catalytic domain. This domain, in the C-terminal region, contains the elements required for catalytic activity (the copper binding site, tyrosyl and lysyl residues that contribute to carbonyl cofactor and 10 cysteine residues.) See Thomassin et al. The Pro-regions of lysyl oxidase and lysyl oxidase-like are required for deposition onto elastic fibers, " J. Biol. Chem. 2005 Dec 30; 280 (52): 42848-55 for more details.

Antibodies can bind both full-length and / or processed LOXL2. Antibodies or antigen-binding fragments thereof are provided herein which bind to LOXL2 as defined in the claims. Anti-LOXL2 antibodies and antigen-binding fragments thereof which bind to and / or inhibit LOXL2 have use in the purification, diagnostic and therapeutic methods described herein. Also provided herein are antibodies or antigen-binding fragments which compete with or specifically bind to any of the antigen-binding antibodies or fragments of the same precedents for binding to LOXL2.

Any such antigen-binding antibody or fragments may specifically bind to LOXL2 with a binding affinity of at least 2.5, 10, 50, 100, 500 or 1000-fold greater than at least one of LOX, LOXL1, LOXL3 or LOXL4.

In one embodiment, an antibody or antigen-binding fragment thereof, described herein specifically binds to the SRX3-4 region of L0XL2 and thereby binds both full-length and processed L0XL2. In one aspect, both full-length and processed L0XL2 are active forms of the enzyme. An antibody may, for example, specifically bind to an epitope having an amino acid sequence shown as SEQ. Such antibodies may serve as a partial inhibitor of enzymatic activity in vitro, inhibiting approximately half of the enzymatic activity against a 1,5-diaminopentane substrate with an apparent IC50 of 20-30 nM. Such antibodies may serve as non-competitive inhibitors.

When humanizing antibodies, simultaneous incorporation of all FR and / or CDR encoding nucleic acids and all selected amino acid positional changes can be achieved by a variety of methods known to those skilled in the art, including for example, recombinant and chemical synthesis. For example, simultaneous incorporation can be achieved, for example, by chemically synthesizing the nucleotide sequence for the acceptor variable region, fused together with the donor CDR encoding nucleic acids and incorporating at the selected positions to harbor variable amino acid residues a plurality of amino acid codons correspondents. Antibodies and antigen-binding fragments thereof are provided herein which bind to L0XL2. Antibodies and antigen-binding fragments thereof that bind to L0XL2 may inhibit (partially or completely) or manage / treat (partially or completely) symptoms associated with and / or caused by aberrant expression of L0XL2. The application also provides cell lines which can be used to produce the antibodies, methods for producing the cell lines, methods for expressing the antigen-binding antibodies or fragments and purifying them.

One will recognize that antibodies and antigen-binding fragments thereof that specifically bind to L0XL2 generated using the methods described herein may be tested using the assays provided herein or known in the art for the ability to bind L0XL2 (e.g., ELISA) as well as for affinity (eg Biacore or Surface Plasma Resonance).

Humanized versions of anti-L0XL2 antibodies have one or more of the following characteristics: retention of the inhibitory function of murine monoclonal antibodies, binding affinity equivalent or increased with a slow dissociation rate (e.g., 0.1-1 nM Kd), binding full-length and / or processed LOXL2, partial noncompetitive inhibition of enzyme activity, equivalent or higher Ic50 (e.g., about 30 nM), inhibitory activity in cell-based migration / invasion assays, inhibition of an EMT-like change LOXL2-induced secretion in conditioned media of tumor cells, binding to matrix-associated LOXL2 generated by live human tumor cells, cross-reactivity of human LOXL2 binding to murine LOXL2, therapeutic efficacy (e.g., partial or reduction in size and / or symptoms of the tumor), reduced toxicity and reduced immunogenicity. Humanized antibodies that bind to hLOXL2 and humanized antibodies that bind to hLOXL2 and LOXL2 (murine LOXL2) are provided herein. In one aspect, the humanized antibodies are non-competitive inhibitors.

In one embodiment, a humanized anti-LOXL2 antibody has a VH chain with an amino acid sequence of SEQ. ID No. 25, SEQ. ID No. 26, SEQ. SEQ ID NO: 27 or SEQ. ID No 28. One would understand that conservative amino acid modifications can be made using the methods described herein in one or more CDR regions or structure for maturation of antibody affinity. Antibodies modified by such methods may be tested for function using any of the assays described herein or known in the art.

In another embodiment, a humanized anti-LOXL2 antibody has a VL chain with an amino acid sequence of SEQ. SEQ ID NO: 30, SEQ. SEQ ID NO: 31 or SEQ. 32. One would appreciate that conservative amino acid modifications can be made using the methods described herein in one or more CDR regions or structure for maturation of antibody affinity. Antibodies modified by such methods may be tested for function using any of the assays described herein or known in the art. There is provided herein a humanized antibody, or antigen-binding fragment thereof, which binds to L0XL2, comprising a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region comprises: (i) a heavy chain FR1 with the amino acid sequence of SEQ. ID No. 33 or the amino acid sequence of SEQ. ID No. 33, but by one or more substitutions selected from the group consisting of: (a) a glutamine (Q) substitution by valine (V) or a conservative substitution thereof at position 24; (b) a leucine (L) substitution per valine (V) or a conservative substitution thereof at position 30; (c) a substitution of valine (V) for lysine (K) or a conservative substitution thereof at position 31; (d) replacing arginine (R) with lysine (K) or a conservative substitution thereof at position 32; and (e) a substitution of threonine (T) for alanine (A) or a conservative substitution thereof at position 35; and a deletion of amino acid residues 1-19; (ii) a heavy chain FR2 with the amino acid sequence of SEQ. ID No. 34 or the amino acid sequence of SEQ. ID No. 34, but by one or more substitutions selected from the group consisting of: (a) a lysine (K) substitution by arginine (R) or a conservative substitution thereof at the 3-position; (b) an arginine (R) substitution by alanine (A) or a conservative substitution thereof at the 5-position, and (iii) a FR3 heavy chain having the amino acid sequence of SEQ. ID No. 35 or the amino acid sequence of SEQ. ID No. 35, but by one or more substitutions selected from the group consisting of: (a) a lysine (K) substitution by arginine (R) or a conservative substitution thereof at the 1-position; (b) a substitution of alanine (A) for valine (V) or a conservative substitution thereof at the 2-position; (c) a replacement of leucine (L) by isoleucine (I) or a conservative substitution thereof at the 4-position; (d) a serine (S) substitution for threonine (T) or a conservative substitution thereof at the 10-position; (e) a glutamine (Q) substitution by glutamic acid (E) or a conservative substitution thereof at position 16; (f) a replacement of threonine (T) by arginine (R) or a conservative substitution thereof at position 21; (g) replacing aspartic acid (D) with glutamic acid (E) or a conservative substitution thereof at position 23; (h) a serine (S) substitution for threonine (T) or a conservative substitution thereof at position 25; and (i) a substitution of phenylalanine (F) for tyrosine (Y) or a conservative substitution thereof at position 29; and (iv) a FR4 heavy chain having the amino acid sequence of SEQ. ID No. 36 or the amino acid sequence of SEQ. 36, but by a lysine (K) substitution for valine (V) or a conservative substitution thereof at position 7, and wherein said light chain variable region comprises: (i) a light chain FR1 with amino acid sequence of SEQ. ID No. 49 or the amino acid sequence of SEQ. ID No. 49, but by one or more substitutions selected from the group consisting of: (a) a substitution of alanine (A) with threonine (T) or a conservative substitution thereof at position 27; (b) a substitution of alanine (A) for proline (P) or a conservative substitution thereof at position 28; (c) a proline substitution (P) for leucine (L) or a conservative substitution thereof at position 29; (d) a substitution of valine (V) for leucine (L) or a conservative substitution thereof at position 31; (e) a substitution of glutamic acid (E) for glutamine (Q) or a conservative substitution thereof at position 37; (d) a substitution of serine (S) by proline (P) or a conservative substitution thereof at position 38; (f) a substitution of valine (V) for alanine (A) or a conservative substitution thereof at position 39; and a deletion of amino acid residues 1-20; (ii) an FR2 light chain with the amino acid sequence of SEQ. ID No. 50 or the amino acid sequence of SEQ. (A) a phenylalanine (F) substitution by tyrosine (Y) or a conservative substitution thereof in the 2-position; and (b) a substitution of arginine (R) for lysine (K) or a conservative substitution thereof at position 5; (iii) an FR3 light chain with the amino acid sequence of SEQ. ID No. 51 or the amino acid sequence of SEQ. ID 51, but by one or more substitutions selected from the group consisting of: (a) a substitution of alanine (A) with aspartic acid (D) or a conservative substitution thereof at the 14-position; and (b) a substitution of arginine (R) for lysine (K) or a conservative substitution thereof at position 18; and (iv) an FR4 light chain with the amino acid sequence of SEQ. ID No. 52 or the amino acid sequence of SEQ. ID No. 52, but by a leucine (L) substitution for valine (V) or a conservative substitution thereof at the 7-position;

In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a FR1 heavy chain variable region with an amino acid sequence, as set forth in SEQ. ID No. 33, 37 or 44; a variable region of the heavy chain FR2 with an amino acid sequence, as set forth in SEQ. ID No. 34, 38 or 45; a FR3 heavy chain variable region with an amino acid sequence, as set forth in SEQ. ID No. 35, 39, 46, 47 or 48; a FR4 heavy chain variable region with an amino acid sequence, as set forth in SEQ. ID No. 36 or 40; a variable region of the FR1 light chain with an amino acid sequence, as set forth in SEQ. ID No. 49 or 53; a variable region of the FR2 light chain with an amino acid sequence, as set forth in SEQ. ID No. 50, 54 or 60; a variable region of the FR3 light chain with an amino acid sequence, as set forth in SEQ. ID No. 51, 55 or 61; and a FR4 light chain variable region with an amino acid sequence, as set forth in SEQ. ID No. 52 or 59.

Covered within the scope of the present application are heavy variable chains and light variable chains which are at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or even 100% identical to the variable chains heavy and light variable chains described herein.

Conservative substitutions are minor modifications of these nucleotide sequences and / or amino acids are intended to be included as heavy and light chains encoding nucleic acids and their functional fragments.

Such minor modifications include, for example, those that do not alter the amino acid sequence encoded due to degeneracy of the genetic code, as well as those that result in only a conservative substitution of the encoded amino acid sequence or those that do not substantially alter the binding capacity of the antibody. Conservative substitutions of encoded amino acids include, for example, amino acids belonging to the following groups: (1) non-polar amino acids (Gly, Ala, Val, Leu and Ile); (2) neutral polar amino acids (Cys, Met, Ser, Thr, Asn and Gin); (3) acidic polar amino acids (Asp and Glu); (4) basic polar amino acids (Lys, Arg and His); and (5) aromatic amino acids (Phe, Trp, Tyr and His). Other minor modifications are included in the nucleic acids encoding the heavy and light chain polypeptides of the invention as long as the encoded nucleic acids or polypeptides retain some or all of their function as described herein and which have use in the methods described herein. Nonconservative substitutions are those that are not identified as conservative substitutions. Using the methods described herein, a person can check whether it would be possible to replace a non-conservative amino acid residue with an amino acid residue structure and to test the function of the modified antibody using the assays described elsewhere herein.

Heavy variable chains and modified light variable chains can be classified in connection with binding and activity using methods known in the art and described herein.

A substantial portion of a variable domain will include three CDR regions, along with their intervening framework regions. The moiety may also comprise at least about 50% of either or both of the first and fourth framework regions, 50% corresponding to 50% of the C-terminal of the first framework region and 50% of the N-terminal of the fourth framework region. Additional residues at the N-terminal or C-terminal end of the substantial part of the variable domain may be those not normally associated with naturally occurring variable domain regions. For example, the construction of humanized anti-L0XL2 antibodies and antigen-binding fragments described herein made by recombinant DNA techniques may result in the introduction of N- or C-terminal residues encoded by binders introduced to facilitate cloning or other steps of manipulation . Other steps of manipulation include the introduction of linkers to join variable domains to additional protein sequences including immunoglobulin heavy chains, other variable domains (e.g., in the production of diabodies), or protein labels, as discussed in more detail below.

Antibodies encompassed by the present application, including, for example, those with heavy or light variable chains having at least 50% identity with those described herein may be evaluated for anti-LOX or anti-LOXL2 activity. There is provided herein a method of identifying an antibody that inhibits metastatic tumor cell growth, comprising contacting L0XL2 or a cell expressing L0XL2 with a candidate antibody; and determining the expression or activity of L0XL2, whereby the candidate antibody that reduces L0XL2 expression or activity compared to the expression or activity of the detected in the absence of the antibody is identified as the compound that inhibits the metastatic tumor cell growth. In particular embodiments, the antibody is contacted with L0XL2 or a cell expressing L0XL2 under hypoxic conditions. In one aspect, the antibodies described herein may be uncompetitive inhibitors.

Also provided herein is a method of identifying an antibody that enhances the efficacy of chemotherapeutic agents, comprising contacting L0XL2 or a cell expressing L0XL2 with a candidate antibody; whereby the candidate antibody that reduces the expression or activity of L0XL2 compared to the expression or activity of the detected in the absence of the antibody is identified as the antibody that enhances the efficacy of the chemotherapeutic agents in inhibiting or reducing metastatic tumor growth.

Any suitable source of L0XL2 may be employed as an antibody target in the present method. The enzyme may be derivatized, isolated or recombinantly produced from any source known in the art, including yeast, microbial and mammalian, which will allow the generation of a suitable product which may generate a detectable reagent or will be biologically active in a suitable assay . The enzymatic activity of L0XL2 can be evaluated by any suitable method described herein or known in the art. Exemplary methods of assessing L0XL2 activity include those of Trackman et al. , Anal. Biochem. 113: 336-342 (1981); Kagan, et al., Methods Enzymol. 82A: 637-49 (1982); Palamakumbura et al. , Anal. Biochem. 300: 245-51 (2002); Albini et al. , Cancer Res. 47: 3239-45 (1987); Kamath et al, Cancer Res. 61: 5933-40 (2001); U.S. Patent No. 4,997,854; and U.S. Patent Application No. 2004/0248871. For example, enzymatic activity can be assessed by detecting and / or quantifying "lysyl oxidase biproducts," " such as H2O2 production; collagen pyridinium residues, ammonia production; production of aldehyde product; oxidation of lysyl, deoxypyridinoline (Dpd) - discussed above. It is also possible to detect and quantify the ability of in vitro cell invasion, adhesion and cell growth in vitro, and in vivo metastatic growth. In vivo models include, but are not limited to, suitable syngeneic models, human tumor xenografts models, orthotopic models, metastatic models, transgenic models, and knockout gene models (see, for example, Teicher, Tumors Models in Cancer Research (Humana Press 2001)).

Hypoxic conditions can be induced or occur naturally. Hypoxic areas often occur within the solid tumor. Hypoxia can also be induced in vivo, particularly in experimental animal models, using decreased or cessation of arterial blood flow to the tumor or administration of vasoconstrictor compounds. See, for example, U.S. Patent No. 5,646,185. Exemplary vasoconstricting compounds include direct and indirect adrenergic agonists, such as norepinephrine, epinephrine, phenylephrine and cocaine. The presence of a hypoxic region in a solid tumor present in a subject can be observed by a number of methods currently known in the art, including nuclear magnetic resonance (NMR) and p02 oxygen electrode histography. Such methods may be used in the context of the present invention (as described below) to identify target regions of hypoxic treatment and to guide the administration of treatment compositions to such regions. Hypoxic conditions, in vitro, may be induced using any suitable method. For example, the cells may be maintained under anoxic conditions (<0.1% O 2) at 37øC in an anaerobic chamber or under hypoxic conditions (1 to 2% O 2) at 37øC in a modular incubator chamber filled with 5% CO2 and 1 to 2% O2 balanced with N2. See, for example, Erler et al., Mol. Cell. Biol. 24: 2875-89 (2004).

LOXL2 enzymes or LOXL2 expressing cells may be contacted with a compound (for example, a LOX / LOXL inhibitor, such as an antibody) in any suitable manner for any suitable amount of time. For regions of the tumor that are accessible for hypodermic delivery of the agent, it may be desirable to inject the compound directly into the hypoxic region. The cells may be contacted with the compound more than once during incubation or treatment. Typically, the dose required for an antibody is in the range of about 1 micro-g / ml to 1000 micro-g / ml, more typically in the range of about 100 pg / ml to about 800 pg / ml. The exact dose can be readily determined from in vitro cell cultures and exposure of the cell to various dosages of the compound. Typically, the amount of time the cell is contacted with the compound is about 5 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 4 hours, about 12 hours, about 36 hours , about 48 hours to about 3 days or more, even indefinitely, more typically for about 24 hours. For in vitro invasion assays, any suitable matrix may be used. In one embodiment, the matrix is Matrigel ™ matrix of reconstituted basement membrane (BD Sciences). Classification methods may also include a step of measuring FAK levels. As described below, FAK (focal adhesion kinase [pl25FAK]) is activated as part of the cellular motile process. When LOX is inhibited, FAK phosphorylation is not increased under hypoxic conditions. In a compound classification assay, a secondary step may include the detection of phospho-FAK levels both with and without the addition of the test antibody. An antibody inhibitor test will also reduce phospho-FAK levels.

An antibody is an inhibitor of the biological expression or activity of LOXL2 when the antibody reduces the expression or activity of LOXL2 relative to that observed in the absence of the antibody. In one embodiment, an antibody is a LOXL2 inhibitor when it reduces the incidence of metastases over that observed in the absence of the antibody and, in additional tests, inhibits metastatic tumor growth. In one aspect, the antibodies described herein are non-competitive inhibitors. Inhibition of the tumor can be quantified using any convenient method of measurement. Incidence of metastases can be assessed by examining the relative spread (e.g., number of organ systems involved) and relative tumor load at these sites. Metastatic growth can be verified by microscopic or macroscopic analysis, as appropriate. Tumor metastases can be reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some embodiments, the antibody may be evaluated relative to other antibodies or compounds that have no impact on the biological expression or activity of LOX or LOXL2. Test antibodies can be administered at the time of tumor inoculation, after establishment of primary tumor growth, or after establishment of local and / or distant metastases. Single or multiple administration of the test antibody can be done using any convenient mode of administration including, but not limited to, intravenous, intraperitoneal, intratumoral, subcutaneous and intradermal.

Any suitable cell expressing LOXL2 may be employed with the present methods. As used herein, the term &quot; cell &quot; includes a biological cell (e.g., CHO, HeLa, etc.). The cell may be human or non-human. The cell may be isolated fresh (i.e., primary) or derived from a short-term or long-term established cell line. Exemplary biological cell lines include MDA-MB 231 human breast cancer cells, MDA-MB 435 human breast cancer cells, U-87 MG glioma, squamous cell carcinoma SCL1, CEM, HeLa epithelial carcinoma and Chinese hamster ovary cells ( CHO). Such cell lines are described, for example, in the Cell Lines Catalog of the American Type Culture Collection (ATCC, Rockville, MD).

A cell may express the L0XL2 or its promoter endogenously or exogenously (for example, as a result of stable gene transfer). Endogenous expression by a cell, as provided herein, may result from the constitutive or induced expression of endogenous genes. Endogenous expression by a cell as provided herein may result from the introduction of the nucleic acid sequences encoding either L0XL2 or a biologically active fragment thereof, or nucleic acid sequence of the LOXL2 promoter promoter. The transformation may be accomplished using viral vectors, calcium phosphate, DEAE-dextran, electroporation, biolistics, cationic lipid reagents or any other convenient technique known in the art. The manner of transformer useful in the present invention is conventional and is exemplified in Current Protocols in Molecular Biology (Ausubel, et al., Eds., 2000). Exogenous expression of lysyl oxidase or its promoter may be transient, stable or some combination thereof. Exogenous expression of the enzyme can be achieved using constitutive promoters, for example, SV40, CMV and the like, and inducible promoters known in the art. Suitable promoters are those that will work in the cell of interest.

The methods described herein are non-limiting and any other methods known in the art can also be used to test the activity of anti-LOXL2 antibodies. Further assays are described below in the Examples.

It may be necessary at times to introduce a non-structured polypeptide linker region between a label of the present invention and portions of the antibodies. The linker may facilitate enhanced flexibility and / or reduce steric hindrance between any two fragments. The linker may also facilitate the occurrence of appropriate folding of each fragment. The linker may be of natural origin, such as a sequence determined to exist in a random spiral between two domains of a protein. An exemplary linker sequence is the linker found between the C-terminal and N-terminal domains of the RNA polymerase subunit. Other examples of naturally occurring linkers include the linkers found in the 1CI and LexA proteins.

Within the linker, the amino acid sequence may be varied based on the preferred linker characteristics, as determined empirically or as disclosed by modeling. Considerations in choosing a linker include the flexibility of the linker, charge of the linker and presence of some amino acids of the linker in the naturally occurring subunits. The linker may also be designed such that the residues of the linker contact the DNA, thereby influencing binding affinity or specificity, or to interact with other proteins. In some cases, particularly where it is necessary to encompass a longer distance between subunits or when the domains have to be maintained in a particular configuration, the linker may optionally contain an additional folded domain.

In some embodiments, it is preferred that the design of a linker involves an array of domains that requires the linker to span a relatively short distance, preferably less than about 10 Angstroms (A). However, in certain embodiments, the binders comprise a distance of up to about 50 A.

Antibodies are provided herein so that they are conjugated or bound to therapeutic and / or tomography / detectable fractions. Methods for conjugating or linking antibodies are well known in the art. Associations between antibodies and labels include any means known in the art including, but not limited to, covalent and non-covalent interactions.

In a non-limiting embodiment, the antibodies may be associated with a toxin, a radionuclide, an iron related compound, a dye, a CT scan reagent, a fluorescent label or a chemotherapeutic agent that would be toxic when delivered to a cancer cell.

Alternatively, the antibodies may be associated with a detectable label, such as a radionuclide, iron-related compound, a dye, a CT scan reagent, or a fluorescent agent for immunizing target antigens.

Non-limiting examples of radiolabels include, for example, 32P, 33P, 43F, 57Co, 64Cu, 67Ga, 67Cu, 68Ga, 71Ge, 75Br, 76Br, 77Br, 77As, 77Br, 81Rb / 81MKr, 97MSr, 90Y, 97Ru, 99Tc, 100Pd, 101Rh, 103Rb, 105Rh, 109Pd, 411Ag, 411In, 113ln, 119Sb, 121Sn, 1231 1251, 127Cs, 128Ba, 129Cs, 1311 131Cs, 143Pr, 153Sm, 161Tb, 166Ho, 169Eu, 177Lu, 186Re, 188Re, 189Re, 1910s 193Pt, 194Bi, 197Hg, 199Au, 203Pb 211At, 212Pb, 1212Bi and 213Bi.

Non-limiting examples of toxins include, for example, diphtheria A chain, non-active diphtheria toxin binding fragments, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, alpha-sarcine, Aleurites fordii proteins, diatin proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcina, crotina, Sapaonaria officinalis inhibitor, gelonin, mitogelin, restrictocine, fenomycin, enomycin, trichothecenes, Clostridium perfringens phospholipase C (PLC), bovine pancreatic ribonuclease (BPR), antiviral protein (PAP), abrin, cobra venom factor (FVC), gelonin (GEL), saporin (SAP) viscumina.

Non-limiting examples of iron-related compounds include, for example, magnetic iron oxide particles, ferrous or ferrous particles, Fe203 and Fe304. Iron-related compounds and methods of labeling polypeptides, proteins and peptides can be found, for example, in U.S. Patents Nos. 4 101 435 and 4 452 773 and published applications U.S. 20020064502 and 20020136693.

In certain embodiments, the subject antibodies may be covalently or non-covalently attached to a cytotoxin or other cell proliferation inhibiting compound, in order to localize the delivery of that agent to a tumor cell. For example, the agent may be selected from the group consisting of agents, enzyme inhibitors, proliferation inhibitors, lytic agents, DNA or RNA synthesis inhibitors, membrane permeability modifiers, DNA metabolites, dichloroethylsulfide derivatives, protein production, ribosome inhibitors, apoptosis inducers and neurotoxins.

In certain embodiments, the subject antibodies may be attached to a useful agent in tumor tomography. Such agents include: metals; metal chelators; lanthanides; lanthanide chelants; radiometais; radiometa chelators; positron emitting cores; microbubbles (for ultrasound); liposomes; microencapsulated molecules in liposomes or nanospheres; monocrystalline iron oxide nanocomposites; screening agents for magnetic resonance tomography; absorbing, reflecting and / or light scattering agents; colloidal particles; fluorophores, such as near-infrared fluorophores. In many embodiments, such secondary fraction / functionality will be relatively large, for example at least 25 amu in size and in many instances may be at least 50, 100 or 250 amu in size.

In certain embodiments, the secondary functionality is a chelated fraction for chelating a metal, for example, a chelator to a radiometal or paramagnetic ion. In additional embodiments, it is a chelator for a radionuclide useful for radiotherapeutic or tomography procedures.

Radionuclides useful in the present invention include gamma emitters, positron emitters, Auger electron emitters, X-ray emitters and fluorescence emitters with preferred beta or alpha emitters for therapeutic use. Examples of radionuclides useful as toxins in radiation therapy include: 32P, 33P, 43K 52Fe, 57Co, 64Ca, 67Ga, 67Cu, 68Ga, 71Ge, 75Br, 76Br, 77Br, 77As, 77Br, 81Rb / 81Mr, 87MSr, 90Y, 97Ru , 99Tc, 100Pd, 101Rh, 103Pb, 105Rh, 109Pd, 411Ag, 411In, 113In, 119Sb, 121Sn, 1231, 1251, 127Cs, 128Ba, 129Cs, 1311, 131Cs, 143Pr, 153Sm, 161Tb, 166Ho, 169EU, 177Lu 186Re , 188Re, 189Re, 1910s, 193Pt, 194Ir, 197Hg, 199Au, 203Pb, 211At, 212Pb, 212Bi and 213Bi.

Preferred therapeutic radionuclides include 188Re, 186Re, 203Pb, 212Pi, 212Bi, 109Pd, 64Cu, 67Cu, 90Y, 125I, 131I, 77Br, 211At, 97Ru, 105Rh, 198Au and 199Ag, 166Ho or 177Lu. Conditions under which a chelator will coordinate a metal are described, for example, by Gasnow et al. U.S. Patent Nos. 4,831,175, 4,454,106 and 4,472,509. In the present invention, &quot; radionuclide &quot; and &quot; radio-label &quot; are interchangeable. 99Tc is a particularly attractive radioisotope for diagnostic applications since it is readily available to all departments of nuclear medicine, is inexpensive, provides minimal radiation doses to the patient and has ideal nuclear tomography properties. It has a half-life of six hours which means that it is desirable to rapidly target a labeled antibody with technetium. Accordingly, in certain preferred embodiments, the modified antibodies include a chelating agent for technetium.

In still other embodiments, the secondary functionality may be a radiosensitizing agent, for example, a fraction that increases the sensitivity of cells to radiation. Examples of radiosensitizing agents include nitroimidazoles, metronidazole and misonidazole (see: DeVita, V.T. in Harrison's Principles of Internal Medicine, page 68, McGraw-Hill Book Co., New York, 1983). Modified antibodies comprising a radiosensitizing agent as the active fraction are administered to localize in the target cell. At the time of exposure of the subject to radiation, the radiosensitizer agent is &quot; excited &quot; and causes cell death.

There is a wide range of fractions which can serve as chelators and which can be derived in the antibodies of the present invention. For example, the chelant may be a derivative of 1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA) and 1-p-isothiocyanato-benzyl-methyl-diethylenetriaminepentaacetic acid (ITC -MX). These chelators typically have side chain groups by which the chelator can be used for attachment to subject antagonists. Such groups include, for example, benzylisothiocyanate, whereby DOTA, DTPA or EDTA may be attached to, for example, an amine group.

In one embodiment, the chelated fraction is a chelated fraction &quot; NxSy &quot;. As defined herein, &quot; chelated NxSy &quot; include bifunctional chelators which are capable of coordinately bonding a metal or radiometal and, preferably, have N2S2 or N3S nuclei. Exemplary NxSy chelates are described, for example, in Fritzberg et al. (1998) PNAS 85: 4024-29; and Weber et al. (1990) Chem. 1: 431-37; and in the references cited therein.

Jacobsen et al. (PCT application WO 98/12156) provide methods and compositions, i.e., synthetic fractions of binding fractions, for identifying compounds which bind to the metal atom. The approach described in that publication can be used to identify binding fractions which may subsequently be added to the antibodies to derive the modified antibodies.

A frequently encountered problem with the use of conjugated proteins in radiodiagnostic applications is a potentially dangerous accumulation of radio-labeled fractions in the kidney. When the conjugate is formed using a labile acid or basic linker, cleavage of the radioactive chelate of the protein may advantageously occur. If the chelate has a relatively low molecular weight, as most of the subject modified antibodies, antigen-binding fragments and peptides are expected to be, is not retained in the kidney and is excreted in the urine, thereby reducing kidney exposure to radioactivity. However, on occasions, it may be advantageous to use acidic or basic labile ligands for the same reasons as those used in labeled proteins.

Accordingly, certain of the subject labeled / modified antibodies can be synthesized, by methods conventional in the art, to provide reactive functional groups which can form acid labile bonds with, for example, a carbonyl group of the ligand. Examples of suitable acid labile bonds include hydrazone and thiosemicarbazone functions. These are formed by reacting the chelated oxidized carbhydrate with hydrazide, thiosemicarbazide and functions, respectively.

Alternatively, basic cleavage may be used which was used for enhanced elimination of the kidney radio label. See, for example, Weber et al. 1990 Bioconj. Chem. 1: 431. Binding of a bifunctional chelate to an antibody through a hydrazide linkage may incorporate base-sensitive ester fractions on a linker spacer arm. Such an ester-containing linker moiety is exemplified by ethylene glycolbis (succinimidyl succinate), (EGS, available from Pierce Chemical Co., Rockford, Ill.), Which has two terminal N-hydroxysuccinimide (NHS) ester derivatives of two acid units 1 , 4-dibutyric acid, each of which is bound to a single fraction of ethylene glycol by two alkyl esters. An NHS ester can be substituted with a suitable amine-containing BFC (e.g., 2-aminobenzyl DTPA), while the other NHS ester is reacted with a limiting amount of hydrazine. The resulting hydrazide is used to bind to the antagonists, forming a BFC-binding linkage containing two alkyl ester functions. Such a conjugate is stable at physiological pH, but rapidly divided at basic pH.

Antibodies labeled by radioisotope chelation are subject to radiation induced scission of the chelator and loss of radioisotope by dissociation of the coordination complex. In some instances, the metal dissociated from the complex may be re-complexed, providing for faster elimination of non-specifically localized isotopes and, therefore, lower toxicity to non-target tissues. For example, chelating compounds, such as EDTA or DTPA, can be infused into patients to provide a group of chelators to bind the released radiometal and facilitate the excretion of free radioisotopes in the urine.

In still other embodiments, the antibodies are attached to a Boron addendum, such as a carborane. Carboranes with carboxyl functions or pendant side chains, for example, can be prepared as is well known in the art. The attachment of such carboranes to amine peptides can be achieved by activating the carboxyl groups of the carboranes and condensing with the amine group to produce the conjugate. Such modified antibodies may be used for neutron capture therapy. The present invention also contemplates the modification of subject dye antagonists, for example, useful in therapeutics and used in conjunction with appropriate non-ionizing radiation. The use of light and porphyrins in the methods of the present invention is also contemplated and their use in cancer therapy has been reviewed by van den Bergh, Chemistry in Britain, 22: 430-437 (1986).

One embodiment of the present invention includes antagonists labeled with a fluorescent label. Common fluorescent labels include, for example, FITC, PE, Texas Red, cytochrome c, etc. Techniques for labeling polypeptides and fragments thereof, such as those provided herein, are well known in the art. The term &quot; anticancer agent &quot; also includes the chemotherapeutic agents described below. The term anticancer agent also includes treatment with a substance that reduces hypoxia in a cell, when such agent is combined with inhibition of LOX. Such a substance may include, for example, p53. See, for example, Matoba et al., &Quot; p53 Regulates Mitochondrial Respiration, &quot; Science June 16, 2006 312: 1650-1653; published online May 24, 2006, and references cited therein. A substance that carries cancer cells into the respiratory tract and away from the glycolytic pathway would be advantageously used with a LOX inhibitor insofar as LOX would not be up-regulated in this case.

Chemotherapeutics useful as active moieties which when conjugated to antagonists thereof of the present invention are delivered specifically to cells are typically small chemical entities produced by chemical synthesis. Chemotherapeutics include cytotoxic and cytostatic drugs. Chemotherapeutics may include those that have other effects on cells, such as reversion of the transformed state to a differentiated state, or those that inhibit cell replication. Examples of known cytotoxic agents useful in the present invention are listed, for example, in Goodman et al., &Quot; The Pharmacological Basis of Therapeutics, &quot; Sixth Edition, A. B. Gilman et al., Eds./Macmillan Publishing Co. New York, 1980. These include taxanes, such as paclitaxel and docetaxel; nitrogen such as mechlorethamine, melphalan, uracil mustard and chlorambucil; ethylenimine derivatives, such as thiotepa; alkyl sulfonates, such as busulfan; nitrosoureas, such as lomustine, semustine and streptozocin; triazenes, such as dacarbazine; folic acid analogues such as methotrexate; pyrimidine analogs, such as fluorouracil, cytarabine and azaribine; purine analogs, such as mercaptopurine and thioguanine; vinca alkaloids, such as vinblastine and vincristine; antibiotics, such as dactinomycin, daunorubicin, doxorubicin and mitomycin; enzymes, such as platinum coordination complexes, such as cisplatin; substituted urea, such as hydroxyurea; methyl hydrazine derivatives, such as procarbazine; adrenocortical suppressants, such as mitotane; hormones and antagonists such as adrenocortisteroids (prednisone), a (hydroxyprogesterone caproate, acetate and megestrol acetate), estrogens (diethylstilbestrol and ethinyl estradiol) and androgens (testosterone propionate and fluoxymesterone). Drugs that interfere with protein synthesis may also be used; such drugs are known to those skilled in the art and include puromycin, cycloheximide and ribonuclease. Most of the chemotherapeutic agents currently in use in the treatment of cancer have functional groups that are directly crosslinkable with an amine or carboxyl group of an agent of the present invention. For example, free amino groups are available in methotrexate, doxorubicin, daunorubicin, cytosinarabinoside, bleomycin, fludarabine and cladribine while free carboxylic acid groups are available in methotrexate, melphalan and chlorambucil.

These functional groups, which are carboxylic acids and free amino acids, are targets for a variety of heterobifunctional and heterobifunctional chemical crosslinking agents that can crosslink these drugs directly to a free amino group of an antagonist.

Chemotherapeutic agents contemplated by the present invention also include other chemotherapeutic drugs that are commercially available. Just to illustrate, the chemotherapeutic may be an inhibitor of chromatin function, an inhibitor, an inhibitor drug, a DNA damaging agent, an antimetabolite (such as folate antagonists, pyrimidine analogues, purine analogues and modified sugar analogs ), an inhibitor of DNA synthesis, an interactive DNA agent (such as an intercalating agent), a DNA repair inhibitor.

Chemotherapeutic agents may be categorized by their mechanism of action, for example, in the following groups: anti-metabolites / anti-cancer agents, such as pyrimidine analogues floxuridine, capecitabine and cytarabine) and purine analogs, folate antagonists and antiproliferative inhibitors related antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine and microtubule such as taxane (paclitaxel, docetaxel), vinblastine, nocodazole, epothilones and navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin , amsacrine, busulfan, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxane, dactinomycin, daunorubicin, doxorubicin, epirubicin, epirubicin, ifosfamide, melphalan, merclorethamine, mitomycin, mitoxantrone, nitrosourea, procarbazine, taxol, taxotere, teniposide, triethylenethiophosoramide and etoposide; as dactinomycin (actinomycin D), gave norrubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mitramycin) and mitomycin; enzymes (L-asparaginase that systematically metabolizes L-asparagine and deprives cells that do not have the ability to synthesize their own asparagine); anti-platelet agents; antiproliferative / antimitotic alkylating agents, such as nitrogen mustards cyclophosphamide and analogs, melphalan, chlorambucil) and (hexamethylmelamine and thiotepa), alkyl nitrosoureas (BCNU) and analogs, streptozocin), trazenos-dacarbazinine (DTIC); antiproliferative / antimitotic antimetabolites, such as folic acid analogues (methotrexate); platinum coordination complexes (cisplatin, oxytoplatinum, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogues (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other thrombin inhibitors); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel; anti-immigration agents; antisecretory agents (breveldine); immunosuppressants tacrolimus sirolimus azathioprine, mycophenolate; compounds (TNP-470, genistein) and growth factor inhibitors (inhibitors of vascular endothelial growth factor, inhibitors of fibroblast growth factor); angiotensin receptor blocker, nitric oxide donors; antisense oligonucleotides; antibodies (trastuzumab, rituximab); cell cycle inhibitors and differentiation inducers (tretinoin); inhibitors of topoisomerase (doxorubicin (adriamycin), daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone and prenisolone); growth factor signal transduction kinase inhibitors; dysfunction inducers, toxins such as cholera tox, ricin, Pseudomonas exotoxin, Bordetella pertussis toxin adenylate cyclase or diphtheria toxin and caspase activators; and chromatin. Preferred dosages of the chemotherapeutic agents are consistent with the presently-dosed dosages.

In addition, other labels, such as biotin followed by streptavidin-alkaline phosphatase (AP), strong-root peroxidase are contemplated by the present invention.

As used herein, the terms &quot; nucleic acid damage &quot; and &quot; nucleic acid damaging agent &quot; refer to any treatment regimen that directly or indirectly damages nucleic acid (e.g., DNA, cDNA, genomic DNA, mRNA, tRNA or rRNA). Examples of such agents include alkylating agents, nitrosoureas, anti-metabolites, plant alkaloids, plant extracts and radioisotopes. Examples of agents also include nucleic acid damaging drugs, for example, 5-fluorouracil (5-FU), capecitabine, Sl (Tegafur, 5-chloro-2,4-dihydroxypyridine and oxonic acid), 5-ethynyluracil, arabinosyl cytosine ( 5-azacytidine (5-AC), 2 ', 2'-difluoro-2'-deoxycytidine (dFdC), purine antimetabolites (mercaptopurine, azathioprine, thioguanine), gemcitabine hydrochloride (Gemzar), pentostatin, allopurinol, 2-fluoro-arabinosyl adenine (2F-ara-A), hydroxyurea, sulfur mustard (bischloroethylsulfide), mechlorethamine, melphalan, chlorambucil, cyclophosphamide, ifosfamide, thiotepa, AZQ, mitomycin C, dianhydrogalactitol, dibromoducitol, (benznitol), nitrosoureas (BCNU, CCNU, 4-methyl CCNU or ACNU), procarbazine, decarbazine, rebeccamycin, anthracyclines such as doxorubicin (ADR), daunorubicin (Cerubicin), idarubicin (Idamycin) and epirubicin (Ellence ), anthracycline analogues such as mitoxantrone, actinomycin D, non-intercalating topoisomerase inhibitors such as epipodophyllotoxins (etoposide = VP16, teniposide = VM-26), podopylotoxin, bleomycin (Bleo), pepleomycin, nucleic acid-adducing compounds including platinum derivatives (eg, cisplatin (CDDP), cisplatin transcript, carboplatin, iproplatin, tetraplatin and oxaliplatin), camptothecin, topotecan, irinotecan (CPT-11), and SN-38. Specific examples of nucleic acid damaging treatments include radiation (for example, focused microwaves, ultraviolet (UV), infrared (IR) or alpha, beta or gamma) and environmental shock (e.g., hyperthermia).

As used herein, the terms &quot; anti-proliferative treatment &quot; and &quot; anti-proliferative agent &quot; means any treatment regimen that directly or indirectly inhibits the proliferation of a cell, virus, bacterium or other unicellular or multicellular organism regardless of whether or not the treatment or agent damages the nucleic acid. Particular examples of anti-proliferative agents are antitumor and anti-viral drugs which inhibit cell proliferation or proliferation or replication of the virus. Examples include cyclophosphamide, azathioprine, cyclosporin A, prednisolone, melphalan, chlorambucil, mechlorethamine, busulfan, methotrexate, 6-mercaptopurine, thioguanine, cytosine arabinoside, taxol, vinblastine, vincristine, doxorubicin, actinomycin D, mithramycin, carmustine, lomustine , semustine, streptozotocin, hydroxyurea, cisplatin, mitotane, procarbazine, dacarbazine and dibromomannitol. Anti-proliferative agents that cause nucleic acid replication errors or inhibit nucleic acid replication are those as nucleoside and nucleotide analogues (e.g., AZT or 5-AZC).

In another embodiment, the anti-LOX antibody may be conjugated to a &quot; receptor &quot; (such as streptavidin) for use in the tumor pre-direction wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a &quot; binding &quot; (e.g., avidin) which is conjugated to a cytotoxic agent (e.g., a radionuclide).

Methodology for labeling polypeptides and fragments thereof including, but not limited to, is that provided herein well known in the art. When the antibodies of the present invention are labeled with a radiolabel or toxin, the antibodies may be prepared as pharmaceutical compositions which are useful for therapeutic treatment of patients where the pharmaceutical compositions are administered to the patient in an effective amount. When the antibodies of the present invention are labeled with a label which can be visualized, the antibodies may be prepared as pharmaceutical compositions which are useful for the diagnosis of patients where the pharmaceutical compositions are administered to the patient in an amount effective for in vivo tomography or wherein the compositions are tested in an in vitro assay. V. Compositions

Each of the antibodies of the present invention may be used as a composition when combined with a pharmaceutically acceptable carrier or excipient. Such pharmaceutical compositions are useful for administration to a subject in vivo or ex vivo and for diagnosing and / or treating a subject with the disclosed antibodies, for example.

Pharmaceutically acceptable carriers are physiologically acceptable to the administered patient and retain the therapeutic properties of the antibodies or peptides with which they are administered. Pharmaceutically acceptable carriers and their formulations are and are generally described in, for example, Remington's Pharmaceutical Sciences (18th Edition, A. Gennaro, Mack Publishing Co., Easton, PA 1990). An exemplary pharmaceutical carrier is physiological saline. The phrase &quot; pharmaceutically acceptable carrier &quot; as used herein means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in the transport or loading of subject antibodies or peptides from the site of administration of one organ, or portion of the body, to another organ, or portion of the body. Each vehicle must be &quot; acceptable &quot; in the sense of being compatible with the other ingredients of the formulation and not deleterious to the patient. Neither the pharmaceutically acceptable carrier should alter the specific activity of the antagonists. Exemplary vehicles and excipients have been provided elsewhere here.

In one aspect, the present invention provides pharmaceutically acceptable or physiologically acceptable compositions including solvents (aqueous or non-aqueous), solutions, emulsions, dispersion media, coatings, delaying or promoting agents, and isotonic, compatible with pharmaceutical administration. Pharmaceutical compositions or pharmaceutical formulations therefore refer to a composition suitable for pharmaceutical use in a subject. The pharmaceutical compositions and formulations include an amount of a compound of the invention, for example, an effective amount of an antagonist of the invention and a pharmaceutically or physiologically acceptable carrier.

Pharmaceutical compositions may be formulated to be compatible with a particular, systemic or local route of administration. Thus, pharmaceutical compositions include carriers, diluents or excipients suitable for administration by various routes.

In a further invention, the compositions of the present invention further comprise a pharmaceutically acceptable additive in order to improve the stability of the antagonist in the composition and / or to control the rate of release of the composition. Pharmaceutically acceptable additives of the present invention do not alter the specific activity of the subject antagonist. A preferred pharmaceutically acceptable additive is a sugar, such as mannitol, sorbitol, glucose, xylitol, trehalose, sorbose, sucrose, galactose, dextran, dextrose, fructose, lactose and mixtures thereof. Pharmaceutically acceptable additives of the present invention may be combined with pharmaceutically acceptable carriers and / or excipients, such as dextrose. Alternatively, preferred pharmaceutically acceptable additive is a surfactant such as polysorbate 20 or polysorbate 80 to increase the stability of the peptide and decrease gelation of the pharmaceutical solution. The surfactant may be added to the composition in an amount of 0.01% to 5% of the solution. The addition of such pharmaceutically acceptable additives enhances the stability and half-life of the composition in storage. Formulation and delivery methods will generally be tailored according to the site and disease to be treated. Exemplary formulations include, but are not limited to, those suitable for parenteral administration, for example intravenous, intraarterial, intramuscular or subcutaneous administration, including micelles encapsulated formulations, liposomes or drug-releasing capsules (active agents incorporated in a biocompatible coating designed for slow release); ingerible formulations; formulations for topical use, such as creams, ointments and gels; and other formulations, such as inhalers, aerosols and vaporizers. The dosage of the compounds of the invention will vary according to the extent and severity of the need for treatment, the activity of the composition administered, the general health of the subject and other considerations well known to the skilled artisan.

Formulations or enteral (oral) formulations may be contained in a tablet (coated or uncoated) capsule (hard or soft), microsphere, emulsion, powder, granule, crystal, suspension, syrup or elixir. Conventional non-toxic solid carriers which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, can be used to prepare solid formulations. Supplementary active compounds (e.g., preservatives, antibacterial, antiviral and antifungal agents) may also be incorporated into the formulations. A liquid formulation may also be used for enteral administration. The carrier may be selected from various oils including petroleum, animal, vegetable or synthetic, for example, peanut oil, soybean oil, mineral oil, sesame oil. Suitable pharmaceutical excipients include, for example, starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol.

Pharmaceutical compositions for enteral, parenteral or transmucosal delivery include, for example, water, saline, phosphate buffered saline, Hank's solution, Ringer's solution, dextrose / saline, and glucose solutions. The formulations may contain auxiliary substances to approximate physiological conditions, such as buffering agents, tonicity adjusting agents, wetting agents, detergents and the like. Additives may also include additional active ingredients, such as bactericidal or stabilizing agents. For example, the solution may contain sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate or triethanolamine oleate. Additional parenteral formulations and methods are described in Bai (1997) J. Neuroimmunol. 80:65, 75; Warren (1997) J. Neurol. Know. 152: 3138; and Tonegawa (1997) J. Exp. Med. 186: 507 515. The parenteral preparation may be enclosed in disposable vials, disposable syringes or multi-dose plastic or glass vials.

Pharmaceutical compositions for intradermal or subcutaneous administration may include a sterile diluent, such as water, saline, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid, glutathione or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates and agents for the adjustment of tonicity, such as sodium chloride or dextrose.

Pharmaceutical compositions for injection include aqueous (where water soluble) solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL ™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol and the like) and suitable mixtures thereof. Fluidity may be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by the use of surfactants. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid and thimerosal. Isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol and sodium chloride may be included in the composition. The resulting solutions may be packaged for use as such or lyophilized; the lyophilized preparation can be further combined with a sterile solution prior to administration.

Pharmaceutically acceptable carriers may contain a compound that stabilizes, augments or retards absorption or elimination. Such compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans; low molecular weight proteins; compositions which reduce the elimination or hydrolysis of peptides; or excipients or other stabilizers and / or buffers. Agents that retard absorption include, for example, aluminum monostearate and gelatin. Detergents may also be used to stabilize or to increase or decrease the absorption of the pharmaceutical composition, including liposomal vehicles. To protect from digestion the compound may be complexed with a composition to render it resistant to enzymatic and acidic hydrolysis or the compound may be complexed in an appropriately resistant vehicle, such as a liposome. Means for protecting compounds from digestion are known in the art (see, for example, Fix (1996) Pharm Res. 13: 1760-1764; Samanen (1996) J. Pharm.Pharmacol.H 48: 119 135) and U.S. Patent No. 5,391,337 which describe lipid compositions for oral delivery of therapeutic agents).

For transmucosal or transdermal administration, suitable penetrants for the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts and fusidic acid derivatives. Transmucosal administration may be through nasal sprays or suppositories (see, for example, Sayani (1996) &quot; Systemic delivery of peptides and proteins across absorptive mucosae &quot; Crit Rev. Ther. Drug Carrier Syst 13:85 184). For transdermal administration, the active compound may be formulated into ointments, salves, gels or creams as generally known in the art. Transdermal delivery systems can also be achieved using patches.

For delivery of inhalation, the pharmaceutical formulation may be administered in the form of an aerosol or spray. For aerosol administration, the formulation may be provided in a finely divided form together with a surfactant and propellant. In another embodiment, the device for delivering the formulation to the respiratory tissue is in which the formulation vaporizes. Other delivery systems known in the art include dry powder aerosols, liquid delivery systems, inhalers, jet air nebulizers and propellant systems (see, for example, Patton (1998) Biotechniques 16: 141-143; Dura Pharmaceuticals, San Diego, Calif., Aradigm, Hayward, Calif., Aerogen, Santa Clara, Calif., And Inhale Therapeutic Systems, San Carlos, Calif.).

Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid. Methods of preparing such formulations are known to those skilled in the art. The materials may also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes directed to cells or tissues using antibodies or viral coat proteins) may also be used as pharmaceutically acceptable carriers. These may be prepared according to the methods known in the art, for example, as described in U.S. Patent Nos. 4 235 871; 4,501,728; 4,522,811; 4,837,028; 6,110,490; 6,096,716; 5,283,175; 5,279,833; Akimaru (1995) Cytokines Mol. Ther. 1: 197-201; Alving (1995) Immunol. Rev. 145: 311; and Szoka (1980) Ann. Rev. Biophys. Bioeng. 9: 467). Biodegradable microspheres or capsules or other biodegradable polymer configurations capable of prolonged delivery of small molecules, including peptides, are known in the art (see, for example, Putney (1998) Nat. Biotechnol., 16: 153-157). Compounds of the invention may be incorporated into micelles (see, for example, Suntres (1994) J. Pharm Pharmacol 46: 238, Woodle (1992) Pharm Res 9: 260-265). Antagonists can be attached to the surface of the monolayer or lipid bilayer. For example, the antagonists may be attached to hydrazide-PEG- (distearoyl-phosphatidine-1) ethanolamine-containing liposomes (see, for example, Zalipsky (1995) Bioconjug. Chem. 6: 705-708). Alternatively, any form of lipid membrane, such as a planar lipid membrane or the cell membrane of an intact cell, for example a red blood cell, may be used. Formulations containing liposomes and lipids may be delivered by any means, including, for example, intravenous, transdermal (see, for example, Vutla (1996) J. Pharm., 85: 58), transmucosal or oral administration.

The compositions of the present invention may be combined with other therapeutic fractions or tomography / diagnostic fractions as provided herein. Therapeutic fractions and / or fractions of tomography can be provided as separate compositions or as a conjugated fraction. Binders for conjugated fractions may be included as needed and have been described elsewhere herein.

The antibodies disclosed herein may also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Know. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Know. USA, 77: 4030 (1980); and U.S. Patents Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.

Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derived phosphatidylethanolamine (PEG-PE). Liposomes are expelled through pore size filters defined to produce liposomes of the desired diameter. Fab 'fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al. , J. Biol. Chem., 257: 286-298 (1982) through a disulfide exchange reaction. A chemotherapeutic agent (such as Doxorubicin) is optionally contained in the liposome. See Gabizon et al., J. National Cancer Inst., 81 (19): 1484 (1989).

Lipid or liposomes may also be used to deliver the anti-LOX antibody or an antibody fragment into the cells. Where antibody fragments are used, the smaller inhibitory fragment that specifically binds to the target protein binding domain may be used. For example, based on the variable region sequences of an antibody, peptide molecules that retain the ability to bind the target protein sequence can be designed. Such peptides may be chemically synthesized and / or produced by recombinant DNA technology. See, for example, Marasco et al., Proc. Natl. Acad. Know. USA, 90: 7889-7893 (1993). The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, including, for example, those having complementary activities that do not adversely affect one another. Alternatively, or in addition, the composition may comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent or growth inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the intended purpose. The active ingredients may also be attached in microcapsules prepared, for example, by coacervation or interfacial polymerization techniques, for example, hydroxymethylcellulose or gelatin microcapsules and poly (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems ( for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.

Formulations for in vivo administration are sterile. Sterilization can be accomplished rapidly by filtration through sterile filtration membranes.

Prolonged-release preparations may be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, for example, films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl methacrylate) or poly (vinylalcohol)), polylactides (US Patent No. 3,773,919), copolymers of L-glutamic acid and γ- L-glutamate, non-degradable ethylene vinyl acetate copolymers, lactic acid-degradable glycolic acid copolymers such as LUPRON DEPOT® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D - (- ) -3-hydroxybutyric acid. While polymers such as ethylene vinyl acetate and lactic acid-glycolic acid allow the release of molecules for more than 100 days, certain hydrogels release proteins for shorter periods of time. When the encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37øC, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be planned for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular SS bond formation through thio-disulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling the moisture content, using appropriate additives and developing specific polymer matrix compositions. Various other pharmaceutical compositions and techniques for their preparation and use will be known to those skilled in the art in light of the present disclosure. For a detailed listing of suitable pharmacological compositions and associated administrative techniques it is possible to reference the teachings detailed herein, which may be further supplemented by texts, such as Remington: The Science and Practice of Pharmacy 20th Edition (Lippincott, Williams & Wilkins 2003).

Pharmaceutical compositions contemplated by the present invention have been described above. In one embodiment of the present invention, the pharmaceutical compositions are formulated to be pyrogen-free so that they are acceptable for administration to human patients. Testing pharmaceutical compositions for pyrogens and preparing pyrogen-free pharmaceutical compositions are well understood by one of ordinary skill in the art.

One embodiment of the present invention contemplates the use of any one of the pharmaceutical compositions of the present invention to make a medicament for treating a disorder of the present invention. Medicaments may be formulated based on the physical characteristics of the patient / subject in need of treatment and may be formulated into single or multiple formulations based on the stage of the cancerous tissue. Medicaments of the present invention may be packaged in a suitable pharmaceutical package with labels appropriate for distribution to hospitals and clinics where the label is for indication of treating a disorder as described herein in a subject. Medications can be packaged as single or multiple units. Instructions for dosing and administration of the pharmaceutical compositions of the present invention may be included with the pharmaceutical packs and kits described below. SAW. Affinity purification

The anti-L0XL2 antibodies described herein are useful for purifying the affinity of L0XL2 from recombinant cell culture, natural sources or tissue (tissue and / or serum) biopsy samples. In this process, antibodies against L0XL2 are immobilized on a suitable carrier, such as Sephadex resin or filter paper, using methods well known in the art. The immobilized antibody is then contacted with a sample containing the L0XL2 to be purified, and thereafter the support is washed with a suitable solvent which will remove substantially all material in the sample except L0XL2, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent which will release the L0XL2 from the antibody. VII. Packages and Kits

One embodiment of the present application includes a pharmaceutical package or kit useful for the methods provided herein. One embodiment of such pharmaceutical packages or kits includes preparations (compositions) of the antagonists, as provided herein.

One aspect of the present invention relates to kits for administering an L0XL2 inhibitor. Another aspect of the present invention relates to kits for carrying out the combined administration of the L0XL2 inhibitor with one or more other therapeutic agents. In one embodiment, the kit comprises an L0XL2 inhibitor formulated in a pharmaceutical carrier or excipient and at least one therapeutic agent which is not said L0XL2 inhibitor, formulated as appropriate, in one or more separate pharmaceutical preparations.

Pharmaceutical packages and kits may additionally include an excipient, a carrier, a buffering agent, a preservative or a stabilizing agent in a pharmaceutical formulation. Each component of the kit may be enclosed in an individual container and all of the various containers may be contained within a single package. The kits of the invention may be designed for storage at ambient or cold temperatures.

In addition, the preparations may contain stabilizers to increase the shelf life of the kits and include, for example, bovine serum albumin (BSA) or other known conventional stabilizers. Where the compositions are lyophilized, the kit may further contain solution preparations for reconstituting the preparations. Acceptable solutions are well known in the art and include, for example, pharmaceutically acceptable phosphate buffered saline (PBS).

In addition, the pharmaceutical packages or kits provided herein may further include any of the other fractions provided herein such as, for example, a chemotherapeutic agent as described elsewhere in more detail.

The pharmaceutical packages and kits of the present invention may further include the components for an assay provided herein, such as, for example, an ELISA assay. Alternatively, kit preparations are used in immunoassays, such as immunohistochemistry to test the tissue biopsy sections of the patient. The pharmaceutical packages and kits of the present invention may further include the components for collecting a sample.

The pharmaceutical packages and kits of the present invention may further include a label specifying, for example, a description of the product, mode of administration and indication of the treatment. Pharmaceutical packs provided herein may include any of the compositions as described herein. The pharmaceutical package may further include a label for preventing, reducing the risk of or treating any of the indications of disease described herein. The term &quot; packaging material &quot; refers to a physical structure that houses the components of the kit. The packaging material can hold the components sterile and may be made from material commonly used for such purposes (eg, paper, corrugated fiber, glass, plastic, razor, canisters, etc.). The packaging label or insert may include appropriate written instructions. Kits of the invention may therefore further include labels or instructions for using the kit components in any method of the invention. A kit can include a compound of the invention in a package, or dispensed together with instructions for administering the compound in a method of the invention.

Instructions may include instructions for practicing any of the methods of the invention described herein including methods of treatment, detection, monitoring or diagnostics. Instructions may additionally include indications of a satisfactory clinical endpoint or any adverse symptoms that may occur or additional information required by regulatory agencies, such as the Administration of Drugs and Foods for use in a human subject.

The instructions may be in a &quot; printed way, &quot; for example, on paper or paperboard inside or affixed to the kit or on a label affixed to the kit or packaging material or attached to a glass vial or tube containing a kit component. Instructions may additionally be included in a computer read medium, such as a disk (floppy disk or hard disk), optical CD such as CD- or DVD-ROM / RAM, magnetic tape, electrical storage media such as RAM and ROM, and hybrids thereof such as magnetic / optical storage media.

The kit compositions of the present invention may be formulated in single or multiple units for a single test or for multiple tests.

In preferred embodiments, the kit preparations are pyrogen-free. Methods for testing the presence of, and / or specific levels of, pyrogens are routine in the art and kits are commercially available for such purpose. There is provided herein a kit for treating a pathological condition associated with L0XL2, containing a composition of an antibody or antigen-binding fragment thereof as described herein and a pharmaceutically acceptable carrier or excipient. The pathological condition associated with L0XL2 may be, for example, a tumor, a metastasis, angiogenesis or fibrosis. In one embodiment, antibodies in such kits may comprise a detectable label, a therapeutic label, or both. In another embodiment, antibodies in such kits can be lyophilized.

Another aspect of the present invention relates to kits for carrying out the combined administration of the L0XL2 inhibitor with other therapeutic compounds. In one embodiment, the kit comprises a LOXL2 inhibitor formulated in a pharmaceutical carrier, and at least one cytotoxic agent, formulated as appropriate, in one or more separate pharmaceutical preparations. VIII. Diagnostic Methods The present invention also provides methods for diagnosing, monitoring, staging or detecting the diseases described above using agents which recognize different forms of L0XL2. For example, as described above, antibodies to different forms of L0XL2, the preproprotein, secreted, mature or active form may be used for these purposes. Methods of diagnosing, monitoring, staging or detecting the diseases described above using agents recognizing different forms of L0XL2 are intended to encompass all of the diseases and indications described herein.

As previously described, the active L0XL2 is divided and may be detected by virtue of its change in molecular weight (immunoblot) or by using antibodies which detect the undivided form vs. divided by L0XL2 together with cell location using various detection methods, such as immunohistochemistry (IHC).

It is thought that the extracellular matrix and conditioned medium should contain proteolytically active active L0XL2, while inactive, undivided L0XI2 should be located intracellularly. Some active, divided L0XI2 may also be detected within the cell as a consequence of extracellular space uptake.

Samples of individuals can be collected and analyzed by determining active or inactive LOX levels. This analysis can be performed prior to initiation of treatment using specific lysyl oxidase therapy to identify tumors with high active L0XL2 expression or activity. Such diagnostic analysis may be performed using any sample, including, but not limited to, cells, proteins or extracts of cell membranes, biological fluids such as sputum, blood, serum, plasma or urine, or biological samples, such as tissue samples, fixed in formalin or frozen tissue sections.

Any suitable method for detecting and analyzing inactive and / or active L0XL2 may be employed. As used herein, the term &quot; sample &quot; refers to a sample of a human, animal or a research sample, for example, a cell, tissue, organ, fluid, gas, aerosol, slurry, colloid or coagulated material. Samples also include, but are not limited to, cell membrane proteins or extracts, biological fluids, such as sputum, blood, serum, plasma or urine or biological samples, such as fixed formalin or tissue sections frozen using the antibodies described on here. The term &quot; sample &quot; may also refer to a cell, tissue, organ or fluid that is harvested fresh from a human or animal, or to a cell, tissue, organ or fluid that is processed or stored. The sample may be tested in vivo, for example, without human or animal removal or may be tested in vitro. The sample may be tested after processing, for example, by histological methods.

Various diagnostic assay techniques known in the art, such as competitive binding assays, direct or indirect sandwich assays, and immunoprecipitation assays conducted in heterogeneous or homogenous phases (Zola, Monoclonal antibodies: A Manual of Techniques, CRC Press, Inc. (1987) pages 147-158). The antibodies used in the diagnostic assays can be labeled with a detectable fraction. The detectable fraction directly or indirectly produces a detectable signal. For example, the detectable moiety may be any of those described herein such as, for example, a radioisotope, such as 3H, 14C, 32P, 355 or 125I, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate (FITC), red Texas, cyanine, photocyan, rhodamine or luciferin, or an enzyme, such as alkaline phosphatase, β-galactosidase or strong-root peroxidase. Any method known in the art for conjugating the antibody to the detectable fraction may be employed, including those methods described by Hunter et al. , Nature, 144: 945 (1962); David et al., Biochemistry, 13: 1014 (1974); Pain et al., J. Immunol. Meth., 40: 219 (1981); and Nygren, J. Histochem. and Cytochem., 30: 407 (1982). There is provided herein a method of diagnosing a pathological condition associated with LOX or LOXL2 which comprises evaluating a level of LOXL2 in a sample of a subject, wherein an Iteration at the L0XL2 level in the sample compared to a reference sample indicates the presence or increase of a tumor or metastasis. In one aspect, the pathological condition associated with LOXL2 is a tumor, a metastasis, angiogenesis or fibrosis. An increase in LOXL2 levels in the sample compared to a reference sample may indicate the presence of a tumor or metastasis or an increase in tumor or metastatic growth. The reference sample may be a sample taken from the subject at an initial time point or a sample from another individual. The level of LOXL2 levels in the sample can be detected by contacting the sample with an antibody or antigen-binding fragment thereof as described herein. In one embodiment, the antibody or antigen-binding fragment thereof is detectably labeled.

In one embodiment, a method is provided for diagnosing carcinogenic metastasis in a subject, comprising: assessing the levels of active LOXL2 or blood activity, whereby a change in levels of active L0XL2 or activity (e.g., gene expression, enzyme activity, etc. .) in blood compared to a reference sample, indicates the presence of metastatic tumor growth. Occasionally, active LOXL2 levels or activities in the blood may be lower than when initially measured, which may indicate that the subject is at increased risk of cancer metastasis; that the cancer metastasized; or that cancer metastasis has increased. The reference sample may be derived from the same subject, taken from the same tumor at a different time point or from another site of the body or from another individual.

In another embodiment, there is provided a method of diagnosing cancer metastasis in a subject having a tumor, comprising: assessing the levels of active L0XL2 or activity in the tumor, whereby a change in active L0XL2 levels or activity in the tumor compared to a reference sample indicates the presence of metastatic tumor growth. On some occasions, active L0XL2 levels or activities in the tumor may be higher than when initially measured, which may indicate that the subject is at increased risk of cancer metastasis; that the cancer metastasized; or that cancer metastasis has increased. The reference sample may be derived from the same subject, taken from the same tumor at a different time point or from another site of the body or from another individual.

Also provided herein is a method of staging tumor growth or metastasis in a subject, comprising evaluating the levels of L0XL2 (e.g., hL0XL2) in a subject tumor, whereby a change in the level of L0XL2 (e.g., gene expression or activity enzymatic) in the tumor compared to a reference sample indicates the presence of metastatic tumor growth. In some instances, L0XL2 levels or activities in the tumor may be higher than when initially measured for the same subject or higher than in a reference sample collected from a normal tissue, which may indicate that the patient is at increased risk of tumor metastasis; that the tumor metastasized; that tumor metastasis increased. The staging of cancers of solid tumors is well known. The TNM system is one of the most commonly used staging systems. This system has been accepted by the International Union Against Cancer (UICC) and the

American Cancer Society (AJCC). Most medical facilities use the TNM system as their primary method of reporting cancer. PDQ®, NCI's comprehensive cancer database, also uses the TNM system. The TNM system, referred to herein as &quot; staging, &quot; is based on the extent of the tumor, the extent of spread to lymph nodes and the presence of metastasis.

Also provided herein is a method of monitoring a response of a subject to a therapy, including an L0XL2 modulator, such as the treatment of cancer, tumors and fibrotic diseases. The method comprises: detecting a change in the level of reactive protein C in the subject upon administration of an L0XL2 modulator to the subject, wherein the change indicates that the LOX modulator or LOXL2 has a therapeutic effect on the subject. A C-reactive protein is an important pharmacodynamic marker for systemic inflammation. It is believed that a reduced level of C-reactive protein (e.g., in the blood sample of the subject) as compared to the previous administration of the L0XL2 inhibitor is indicative of the response of the subject to the therapy using an L0XL2 inhibitor. Measurement of the active L0XL2 levels may take the form of an immunological assay, which detects the presence of an active L0XL2 protein with an antibody to the protein, preferably an antibody that specifically binds to the active L0XL2. Antigen-binding fragments of anti-L0XL2 antibodies can also be used.

Immunoassays can also be used together with laser-induced fluorescence (see, for example, Schmalzing and Nashabeh, Electrophoresis 18: 2184-93 (1997); and Bao, J. Chromatogr., B. Biomed., Sci. 1997) Liposomal immunoassays, such as flow liposomal immunoassays and liposomal immunosensors can also be used to determine active LOX or LOXL levels, according to a method of the invention (Rongen et al., J. Immunol. Immunoassays, such as enzyme-linked immunosorbent assays (ELISAs), may be particularly useful in a method of the invention. A radioimmunoassay may also be useful in determining whether a sample is positive for active LOXL2 or to determine the level of active LOXL2. A radioimmunoassay using, for example, a secondary antibody labeled with iodine-125 may be used.

In addition, it is possible to measure active LOXL2 activity, thus ignoring the amount of inactive enzyme. The enzymatic activity of the active LOXL2 can be measured in a number of ways, using a soluble elastin or lysine soluble collagen labeled as a substrate. Details of an activity assay are given in Royce et al., &Quot; Copper metabolism in mottled mouse mutants. The effect of copper therapy on lysyl oxidase activity in brindled (Mobr) mice, &quot; Biochem J. Feb. 15, 1982; 202 (2): 369-371. An exemplary assay is a chromogenic assay, such as that described by Palamakumbura, et al. &quot; A fluorometric assay for detection of lysyl oxidase enzyme activity in biological samples, &quot; Anal. Biochem. January 15, 2002; 300 (2): 245-51.

In addition to measuring the level of LOXL2 in the blood (or urine), it is possible to measure secondary products of LOXL2 activity. For example, deoxypyridinoline (Dpd) is formed by the enzymatic action of lysyl oxidase on lysine residues. Dpd is released into the circulation as a result of bone osteoclastic degradation. It can not be reused, it is eliminated by the kidney and is excreted unchanged in the urine. Thus, a test based on the Assay BCT Dpd Assay of Immunodiagnostic Systems (IDS) can be used, using a RIA coated tube using an anti-Dpd monoclonal antibody to measure the enzymatic activity.

The anti-LOXL2 antibodies described herein may also be used in the diagnosis of diseases or pathological conditions associated with aberrant collagen metabolism, such as various fibrotic pathological conditions, e.g., pulmonary fibrosis, as well as in proliferative vitreous retinopathy, surgical healing, systemic sclerosis , scleroderma, contraction of wounds, hypertrophic scars, fibromatosis (especially Dupuytren's disease) and keloids. IX. Therapeutic methods

The pharmaceutical formulations according to the present invention may be used to treat a wide variety of diseases and disorders, such as, for example, cancer, metastasis, fibrosis and aberrant angiogenesis. There is provided herein a method of inhibiting L0XL2 by contacting a sample or a cellular tissue with any of the anti-L0XL2 antibodies or antigen-binding fragments thereof as described herein. Binding of said antibody or antigen-binding fragment thereof to L0XL2 inhibits the enzymatic activity of L0XL2.

In any such method, the contacting may occur in vitro, in vivo or ex vivo. Inhibition of L0XL2 may have one or more effects on a subject such as, for example, reduction in tumor growth, reduction in angiogenesis, reduction in fibrotic disease and / or decrease in extracellular matrix formation. Fibrotic diseases include, but are not limited to, hepatic fibrosis, pulmonary fibrosis, renal fibrosis, cardiac fibrosis, and scleroderma. There are provided herein L0XL2 antibodies for use in methods for treating diseases and disorders associated with aberrant expression of or L0XL2. Diseases and disorders include, but are not limited to, tumors (e.g., primary or metastatic), pathological conditions related to angiogenesis and pathological fibrotic conditions.

As used herein, &quot; prevention &quot; refers to prophylaxis, prevention of the onset of symptoms, prevention of the progression of a disease or disorder associated with fibrosis or correlated with L0XL2 activity. As used herein, &quot; treat &quot; or &quot; treatment &quot; means stasis or a delay in the development of the symptoms associated with a disease or disorder described herein. The terms further include improving existing uncontrolled or unwanted symptoms, preventing further symptoms and improving or preventing the underlying metabolic causes of the symptoms. Thus, the terms indicate that a beneficial result has been conferred on a mammalian subject with a disease or symptoms or with the potential to develop such diseases or symptom. A response is achieved when the patient experiences partial or total relief or reduction of signs or symptoms of disease and specifically includes, without limitation, prolongation of survival. Expected progression-free survival times can be measured in months to years, depending on prognostic factors including the number of recurrences, disease stage and other factors. Prolongation of survival includes, without limitation, times of at least 1 month (mo), about at least 2 months (months), at least 3 months, at least 4 months, at least 6 months, about at least 1 year, about at least 2 years, about at least 3 years or more. Global survival can also be measured in months to years. The patient's symptoms may remain static or may subside.

The pharmaceutical compositions of the present invention are administered in therapeutically effective amounts which are effective to produce any desired therapeutic effect at a reasonable benefit / risk ratio applicable to any medical treatment. For administration of the present pharmaceutical compositions to human patients, the pharmaceutical compositions of the present invention may be formulated by the methodology known to one of ordinary skill in the art to be substantially free of pyrogens such that they do not induce an inflammatory response.

As used herein, the term &quot; therapeutically effective amount &quot; or &quot; effective amount &quot; refers to an amount of a therapeutic agent which when administered alone or in combination with another therapeutic agent to a cell, tissue or subject is effective in preventing or ameliorating the disease condition or progression of the disease. A therapeutically effective dose further refers to that amount of the compound sufficient to result in the amelioration of symptoms, for example, treatment, cure, prevention or amelioration of the relevant medical condition or increased rate of treatment, cure, prevention or amelioration of such conditions pathological conditions. When applied to an individual active ingredient administered alone, a therapeutically effective dose refers to that ingredient alone. When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or concurrently. For example, where in vivo administration of an anti-L0XL2 antibody is employed, normal dosage amounts may range from about 10 ng / kg to 100 mg / kg of mammalian body weight or more per day, preferably about 1 kg / day to 50 mg / kg / day, optionally about 100 æg / kg / day to 20 mg / kg / day, 500 æg / kg / day to 10 mg / kg / day or 1 mg / kg / day at 10 mg / kg / day, depending on the route of administration.

An effective response of the present invention is achieved when the patient experiences partial or total relief or reduction of signs or symptoms of the disease and specifically includes, without limitation, prolongation of survival. Expected progression-free survival times can be measured in months to years, depending on prognostic factors including the number of recurrences, disease stage and other factors. Prolongation of survival includes, without limitation, times of at least 1 month (mo), about at least 2 months, at least 3 months, at least 4 months, at least 6 months, at least less 1 year, at least 2 years, at least 3 years or more. Global survival can also be measured in months to years. The patient's symptoms may remain static and the burden of the tumor may not increase.

A physician or veterinarian of ordinary skill in the art can readily determine and prescribe the effective amount (ED50) of the required pharmaceutical composition. For example, the physician or veterinarian may begin by doses of the compounds of the invention employed in the pharmaceutical composition at levels below those necessary to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

As used herein, the term &quot; subject &quot; means mammalian subjects. Exemplary subjects include, but are not limited to, humans, monkeys, dogs, cats, mice, rats, cows, horses, goats and sheep. In some embodiments, the subject has cancer and can be treated with the agent of the present invention as described below.

Regardless of the route of administration selected, the compounds of the present invention which are used in a suitably hydrated form and / or the pharmaceutical compositions of the present invention are formulated into pharmaceutically acceptable dosage forms as described below or by other conventional methods known to those enabled in art.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response to a particular patient, composition and mode of administration without being toxic to the patient. The level of dosage selected will depend on a variety of factors, including the activity of the particular compound of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound to be employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular composition employed, with the age, sex, weight, physical condition, general health and medical history of the patient to be treated, and related factors well known in the medical arts.

In one aspect provided herein, administration of the antibodies results in an improvement in the condition of the subject. In another aspect, administration of the antibodies prevents the condition of the subject from worsening and / or prolongs the survival of the patient. The patient may be a mammal, such as a human or a non-human. Such a patient may be symptomatic or asymptomatic.

The compositions may be administered locally, regionally or systemically by any suitable route provided herein.

In one aspect, the patient's symptoms are improved. The improvement may be manifested as, for example, reduction of pain, reduced tumor size, tumor clearance, prevention of increases in tumor size or progression or disease, prevention of metastasis formation or inhibition of metastatic growth, inhibition of fibrosis , inhibition of angiogenesis or a combination thereof.

If necessary, for cancer treatments, the methods may include surgical removal of the cancer and / or administration of an anti-cancer agent or treatment. Administration of such anti-cancer agent or treatment may coincide with administration of the compositions disclosed herein. Anti-cancer agents have been provided elsewhere here.

In one aspect, administration of any of the antibodies provided herein reduces or eliminates the need for the patient to undergo surgery or treatment with one or more anti-cancer agents or treatments.

Indications that can be treated using the pharmaceutical formulations of the present invention include those involving undesirable or uncontrolled cell proliferation. Such indications include benign tumors, various types of cancers, such as primary tumors and tumor metastasis, restenosis (e.g., coronary, carotid and brain lesions), hematological disorders, abnormal stimulation of endothelial cells (atherosclerosis), damage to body tissue due to surgery, abnormal scarring, abnormal angiogenesis, tissue fibrosis-producing diseases, macular degeneration, glaucoma; age-related macular degeneration (AMD moist and dry AMD), atherosclerosis, rheumatoid arthritis, multiple sclerosis, liver fibrosis, renal fibrosis, pulmonary fibrosis, scleroderma, atherosclerosis and Alzheimer's disease, repetitive motor disorders, disorders of tissue that are not highly and proliferative responses associated with organ transplants. Hepatic fibrosis includes, but is not limited to, cirrhosis and associated pathological conditions such as chronic viral hepatitis, non-alcoholic fatty liver disease (NAFLD), alcoholic steatohepatitis (ASH), non-alcoholic steatohepatitis (NASH), biliary cirrhosis (PBC), biliary cirrhosis and autoimmune hepatitis. Pulmonary fibrosis includes, but is not limited to, idiopathic pulmonary fibrosis (IPF) or cryptogenic fibrosing alveolitis, chronic fibrosing interstitial pneumonia, interstitial lung disease (ILD), diffuse pulmonary parenchymal disease (DPLD), emphysema and chronic obstructive pulmonary disease COPD) and chronic asthma. Cardiac fibrosis includes, but is not limited to, congestive heart failure, cardiomyopathy, and post-acute myocardial infarction defects in cardiac function. Renal fibrosis includes, but is not limited to, diabetic nephropathy, vesicoureteral reflux, tubulointerstitial renal fibrosis; glomerulonephritis or glomerular nephritis, including focal segmental glomerulosclerosis and membranous glomerulonephritis and mesangiocapillar glomerular nephritis.

In general, cells in a benign tumor retain their differentiated characteristics and do not divide in a completely uncontrolled manner. A benign tumor is usually localized and is non-metastatic. Specific types of benign tumors that can be treated using the present invention include hemangiomas, hepatocellular adenoma, cavernous hemangioma, focal nodular hyperplasia, acoustic neuromas, neurofibroma, bile duct adenoma, bile duct cysticoma, fibroma, lipomas, leiomyomas, mesotheliomas, teratomas , myxomas, nodular regenerative hyperplasia, trachoma, pyogenic granulomas, warts, uterine fibroids, thyroid adenomas, adrenocortical adenomas and pituitary adenomas.

In a malignant tumor, the cells become undifferentiated, do not respond to the growth control signals of the body and multiply in an uncontrolled manner. The malignant tumor is invasive and capable of spreading to distant sites (metastasis). Malignant tumors are generally divided into two categories: primary and secondary. Primary tumors arise directly from the tissue in which they are found. A secondary tumor, or metastasis, is a tumor that originates elsewhere on the body, but has now spread to a distant organ. Common pathways for metastasis are direct growth to adjacent structures, spread to the vascular or lymphatic systems, and tracking along tissue planes and body spaces (peritoneal fluid, cerebrospinal fluid, etc.)

Specific types of cancers or malignant tumors, whether primary or secondary, that can be treated using this invention include, but are not limited to, lung cancer (including pulmonary adenocarcinoma, squamous cell carcinoma, large cell carcinoma, bronchioloalveolar carcinoma, carcinoma non-small cell, small cell carcinoma, mesothelioma); breast cancer (including carcinoma, lobular carcinoma, inflammatory breast cancer, clear breast cancer, mucinous carcinoma); colorectal cancer (colon cancer, rectal cancer); anal cancer; pancreatic cancer (including pancreatic adenocarcinoma, islet cell carcinoma, neuroendocrine tumors); prostate cancer; ovarian carcinoma (ovarian epithelial carcinoma or tumor of the epithelial surface-stroma including serous tumor, endometrioid tumor and mucinous cystadenocarcinoma, sex-stromal tumor); hepatic and bile duct carcinoma (including hepatocellular carcinoma, cholangiocarcinoma, hemangioma); oesophageal carcinoma (including esophageal adenocarcinoma and squamous cell carcinoma); non-Hodgkin's lymphoma; bladder carcinoma; uterine carcinoma (including endometrial adenocarcinoma, uterine papillary serous carcinoma, uterine clear cell carcinoma, uterine sarcomas and leiomyosarcomas, mixed mulerian tumors); glioma, glioblastoma, medulablastoma and other brain tumors; renal cancers (including renal cell carcinoma, clear breast cancer, Wilm's tumor); cancer of the head and neck (including squamous cell carcinomas); stomach cancer (stomach adenocarcinoma, gastrointestinal stromal tumor); multiple myeloma; testicular cancer; germ cell tumor; neuroendocrine tumor; cervical cancer; carcinoids of the gastrointestinal tract, breast and other organs; signaling ring cell carcinoma; mesenchymal tumors including sarcomas, fibrosarcomas, hemangioma, angiomatosis, haemangiopericytoma, pseudoangiomatous stromal hyperplasia, myofibroblastoma, fibromatosis, inflammatory myofibroblastic tumor, lipoma, angiolipoma, granular cell tumor, neurofibroma, schwannoma, angiosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, leiomyoma or leiomyosarcoma. The term &quot; metastasis &quot; means the ability of tumor cells to invade the tissues of the host and metastasize to specific, often distant organ sites. As is known, this is the salient feature of lethal tumor growths. The formation of metastasis occurs through a complex series of unique interactions between tumor cells and normal host tissues and cells. In the context of the present invention, the activity of lysyl oxidase is critical in the metastatic growth of tumors, i.e. in the growth of metastases, particularly under hypoxic conditions. Since hypoxic tumors are also the most aggressive and resistant to traditional chemotherapeutic agents, agents that modulate lysyl oxidase expression and / or function provide novel therapeutics against metastatic tumors, particularly chemo-resistant tumors. &quot; Metastasize &quot; is distinguished from invasion. As described in &quot; Understanding Cancer Series: Cancer, &quot; on the website: cancer. gov / cancertopics / understandingcancer / cancer; invasion refers to direct migration and penetration by cancer cells into neighboring tissues.

Hematologic disorders include abnormal growth of blood cells leading to dysplastic changes in blood cells and hematological malignancies such as various leukemias. Examples of hematological disorders include, but are not limited to, acute myeloid leukemia, acute promyelocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, myelodysplastic syndromes, and sickle cell anemia.

Acute myeloid leukemia (AML) is the most common type of acute leukemia that occurs in adults. Several inherited genetic disorders and immunodeficiency states are associated with an increased risk of AML. These include disorders with defects in DNA stability, leading to random chromosome breakage, such as Bloom's syndrome, Fanconi's anemia, Li-Fraumeni kinship syndrome, ataxia-telangiectasia, and X-linked agammaglobulinemia. Acute promyelocytic leukemia (APML) represents a distinct subgroup of AML. This subtype is characterized by promyelocytic blasts containing chromosome translocation 15; 17. This translocation leads to generation of the fusion transcript comprised of the retinoic acid receptor and a PML sequence. Acute lymphoblastic leukemia (ALL) is a heterogeneous disease with distinct clinical features exhibited by several subtypes. Recurrent cytogenetic abnormalities were demonstrated in ALL. The most common cytogenetic anomaly is the translocation 9; 22. The resulting Philadelphia chromosome represents a poor prognosis of the patient. Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder of a pluripotent stem cell. CML is characterized by a specific chromosomal anomaly that involves the translocation of chromosomes 9 and 22, creating the Philadelphia chromosome. Ionizing radiation is associated with the development of CML.

Myelodysplastic syndromes (MDS) are heterogeneous cloned hematopoietic stem cell disorders grouped together because of the presence of dysplastic changes in one or more of the hematopoietic lineages including dysplastic changes in the myeloid, erythroid, and megakaryocytic series. These changes result in cytopenias in one or more of the three lineages. Patients with MDS typically develop complications related to anemia, neutropenia (infections) or thrombocytopenia (bleeding). In general, from about 10% to about 70% of patients with MDS develop acute leukemia.

The administration of LOX2 inhibitors has been found to reduce the size of existing tumors, prevent metastases, and reduce the size of (or even eliminate) existing metastases (see, for example, Moinar et al. (2003) Biochim Biophys. : 220-224). There is provided herein a method of reducing tumor growth in a subject by administering any of the anti-LOX or anti-LOXL2 antibodies or antigen binding fragments thereof as described herein. In one embodiment, a tumor is a primary tumor. In another embodiment, a tumor is a metastatic tumor. The metastatic tumor burden of a subject can be stabilized by administering antibodies as described herein. The tumor in the subject can be reduced by at least 10%, 25%, 50%, 70%, 90%, 95%, or more as compared to the tumor in the subject prior to treatment.

When an antibody or antigen-binding fragment thereof binds specifically to LOXL2, examples of tumors include, but are not limited to, a colon tumor, an oesophageal tumor, a mammary tumor, a prostate tumor, a squamous or a fusocellular carcinoma.

When an antibody or antigen-binding fragment thereof binds specifically to LOX, examples of tumors include, but are not limited to, a mammary tumor, a lung tumor, a renal tumor, a uterine tumor, a liver tumor, or a tumor head and neck. There is provided herein a method of preventing or reducing tumor growth, metastatic tumor growth, in an in vivo subject, comprising administering to a subject in need thereof an effective amount of an inhibitor of LOX or L0XL2 activity; optionally, a pharmaceutically acceptable carrier or excipient, thereby preventing or reducing tumor growth, for example at least 25%, 50%, 75%, 90% or 95%, in the subject treated. A detailed description of compositions suitable for use in the present methods of treatment is given below. Such methods are useful, for example, when the tumor is hypoxic. Hypoxic tumors can be readily identified using methods routinely in the art. See, for example, U.S. Patent No. 5,674,693.

In addition, there is provided herein a method of treating metastasis in a subject with cancer in vivo, comprising administering to a subject in need thereof an effective amount of a LOXL2 inhibitor, thereby inhibiting metastasis, for example in at least 25 %, 50%, 75%, 90% or 95% in the subject treated. In one embodiment, inhibitors specifically inhibit human LOXL2. Antibodies to be used in these methods have been described above. It may be desirable for the antibody to minimize cross-reactions with other members of the LOXL2 families and thus reduce potential adverse side effects due to normal tissue complications and toxicity.

Also provided herein is a method of increasing or potentiating the survival probabilities of a subject with metastatic tumor comprising administering to a subject in need thereof an effective amount of an anti-L0XL2 antibody, thereby enhancing or enhancing the probabilities of survival of the subject treated for a certain period of time, for example at least 10 days, 1 month, 3 months, 6 months, 1 year, 1.5 years, 2 years, 3 years, 4 years, 5 years, 8 years, 10 years or more. The increase in survival of a subject can be defined, for example, as the increase in survival of a preclinical animal model of cancer metastases (e.g., a mouse with metastatic cancer) for a certain period of time, at least 10 days, 1 month, 3 months, 6 months, or 1 year, or at least 2 times, 3 times, 4 times, 5 times, 8 times, or 10 times, more than a control animal model the same type of metastatic cancer) without treatment with the inventive method. Alternatively, the increase in survival of a mammal may also be defined, for example, as the increase in survival of a patient with cancerous metastases for a certain period of time, e.g. at least 10 days, 1 month, 3 months , 6 months, 1 year, 1.5 years, 2 years, 3 years, 4 years, 5 years, 8 years, 10 years or more than one patient with the same type of metastatic cancer but without treatment with the method inventive. The control patient may be on placebo or treated with standard supportive care, such as chemical, biological and / or radiation therapy which does not include the inventive method as a part of the therapy.

Also provided herein is a method of stabilizing the metastatic tumor burden of a subject, comprising administering to a subject in need thereof an effective amount of an anti-LOX antibody or an anti-LOXL2 antibody, thereby stabilizing the charge of metastatic tumor of a subject during a certain period of time, for example for at least 10 days, 1 month, 3 months, 6 months, 1 year, 1.5 years, 2 years, 3 years, 4 years, 5 years, 8 years, 10 years or more. Stabilization of the metastatic tumor burden of a subject can be defined as stabilization of the metastatic tumor burden of a preclinical animal model with metastatic tumor burden (for example, a mouse with metastatic tumor) over a period of time, e.g. For example, for at least 10 days, 1 month, 3 months, 6 months or 1 year longer than a control animal model (having the same type of metastatic tumor) without treatment with the inventive method.

The present methods of treatment also include a method of increasing the efficacy of the chemotherapeutic agents comprising administering to a subject in need thereof an effective amount of an anti-LOXL2 antibody; and optionally, a pharmaceutically acceptable carrier or excipient, thereby enhancing the efficacy of the chemotherapeutic agents (which are described in more detail above). Also contemplated are methods involving the delivery of LOX inhibitor formulations in combination with radiation therapy. Radiation therapy can be used to treat almost all types of solid tumors, including cancers of the brain, breast, cervix, larynx, lung, pancreas, prostate, skin, spine, stomach, uterus or soft tissue sarcomas. Radiation can also be used to treat leukemia and lymphoma (cancers of blood-forming and lymphatic system, respectively). The dose of radiation for each site depends on a number of factors including the type of cancer and whether there are nearby tissues and organs that can be damaged by radiation. The radiation will typically be delivered as X-rays, where the dosage is dependent on the tissue being treated. Radiopharmaceuticals, also known as radionucleotides, may also be used to treat cancer, including thyroid cancer, breast cancer recurrence, and pain caused by the spread of cancer to the bone (bone metastases). Radionuclides were described in more detail below. The subject to be treated or diagnosed by the present methods includes a subject with or at risk of having metastatic tumor growth. In one aspect, a tumor is, for example, lung cancer (including lung adenocarcinoma, squamous cell carcinoma, large cell carcinoma, bronchioloalveolar carcinoma, non-small cell carcinoma, small cell carcinoma, mesothelioma); breast cancer (including carcinoma, lobular carcinoma, inflammatory breast cancer, clear breast cancer, mucinous carcinoma); colorectal cancer (colon cancer, rectal cancer); anal cancer; pancreatic cancer (including pancreatic adenocarcinoma, islet cell carcinoma, neuroendocrine tumors); prostate cancer; ovarian carcinoma (ovarian epithelial carcinoma or tumor of the epithelial surface-stroma including serous tumor, endometrioid tumor and mucinous cystadenocarcinoma, sex-stromal tumor); hepatic and bile duct carcinoma (including hepatocellular carcinoma, cholangiocarcinoma, hemangioma); oesophageal carcinoma (including esophageal adenocarcinoma and squamous cell carcinoma); non-Hodgkin's lymphoma; bladder carcinoma; uterine carcinoma (including endometrial adenocarcinoma, uterine papillary serous carcinoma, uterine clear cell carcinoma, uterine sarcomas and leiomyosarcomas, mixed mulerian tumors); glioma, glioblastoma, medulablastoma and other brain tumors; renal cancers (including renal cell carcinoma, clear breast cancer, Wilm's tumor); cancer of the head and neck (including squamous cell carcinomas); stomach cancer (stomach adenocarcinoma, gastrointestinal stromal tumor); multiple myeloma; testicular cancer; germ cell tumor; neuroendocrine tumor; cervical cancer; carcinoids of the gastrointestinal tract, breast and other organs; signaling ring cell carcinoma; mesenchymal tumors including sarcomas, fibrosarcomas, hemangioma, angiomatosis, hemangiopericytoma, pseudoangiomatous stromal hyperplasia, myofibroblastoma, fibromatosis, inflammatory myofibroblastic tumor, lipoma, angiolipoma, granular cell tumor, neurofibroma, schwannoma, angiosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, leiomyoma or leiomyosarcoma . In a non-limiting embodiment, the tumor is a mammary tumor, a tumor of the pancreas, a lung tumor, a cervical tumor, a tumor of the colon or a tumor of the head and neck. The present invention also provides L0XL2 antibodies for use in a method for preventing or reducing the risk of tumor metastasis in a subject, comprising administering to a subject in need thereof an effective amount of an anti-L0XL2 antibody; and optionally, a pharmaceutically acceptable carrier or excipient, thereby preventing or reducing the risk of tumor metastasis. The inhibitor may be an antibody or an antigen-binding fragment thereof. The subject in need of such prophylactic may be an individual who is genetically predisposed to cancer to run a high risk of developing cancer due to various reasons such as family history of cancer and carcinogenic environment.

Examples of the human genes that are involved in triggering the development of cancer include, but are not limited to, VHL (the Von Hippon Landau gene involved in renal cell carcinoma); P16 / INK4A (involved in lymphoma); E-cadherin (involved in the metastasis of breast, thyroid, gastric cancer); hMLH1 (involved in DNA repair in colon, gastric and endometrial cancer); BRCA1 (from in DNA repair in breast and ovarian cancer); LKB1 (involved in colon and breast cancer); P15 / INK4B (involved in leukemia such as AML and ALL); ER (estrogen receptor, involved in colon cancer, breast and leukemia); 06-MGMT (involved in repairing DNA in brain, colon, lung and lymphoma cancer); GST-pi (involved in breast, prostate and renal cancer); TIMP-3 (tissue metalloprotease, involved in the metastasis of colon, renal and brain cancer); DAPK1 (DAP kinase, involved in apoptosis of B cell lymphoma cells); P73 (involved in apoptosis of lymphoma cells); AR (androgen receptor, involved in prostate cancer); RAR-beta (retinoic acid receptor-beta, involved in prostate cancer); endothelin-B receptor (involved in prostate cancer); Rb (involved in retinoblastoma cell cycle regulation); p53 (a major tumor suppressor gene); P14ARF (involved in cell cycle regulation); RASSFl (involved in signal transduction); APC (involved in signal transduction); Caspase-8 (involved in apoptosis); TERT (involved in senescence); TERC (involved in senescence); TMS-1 (involved in apoptosis); SOCS-1 (involved in hepatocarcinoma growth factor response); PITX2 (hepatocarcinoma breast cancer); MINT1; MINT2; GPR37; SDC4; MY0D1; MDR1; THBS1; PTC1; and pMDR1, as described in Santini et al. (2001) Ann. of Intern. Med. 134: 573-586. Nucleotide sequences from these genes can be retrieved from the website of the National Center for Biotechnology Information (NCBI).

It should be noted that although leukemia is a blood cancer, it can affect other organs, or, indeed, metastasize. In acute leukemias, abnormal cells can collect in the central nervous system, testicles, skin and any other organ in the body. Because leukemia already involves the entire bone marrow in the body and in many cases has spread to other organs such as liver, spleen and lymph nodes, the staging of leukemia depends on other information that reflects the patient's perspective regarding survival. Leukemias include, for example, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML) and hairy cell leukemia (LCH). Different staging systems are used for different types of chronic leukemia. Some types do not have any staging system. Methods of enactment are described in more detail below. Treatment of abnormal cell proliferation due to damage to body tissue during surgery may be possible for a variety of surgical procedures including joint surgery, bowel surgery and keloid healing. Diseases that produce fibrotic tissue include emphysema. Repetitive motor disorders that can be treated using the present invention include carpal tunnel syndrome. An example of cellular proliferative disorders that can be treated using the invention is a bone tumor. There are provided herein L0XL2 antibodies for use in a method for preventing or reducing the risk of abnormal cell proliferation due to damage to body tissue during surgery or a disease that produces fibrotic tissue in a subject comprising administering to a subject in need thereof an effective amount an anti-LOX antibody or an anti-L0XL2 antibody; and optionally, a pharmaceutically acceptable carrier or excipient, thereby preventing or reducing abnormal cell proliferation due to damage to body tissue during surgery. The inhibitor may be an antibody or an antigen-binding fragment thereof. The subject in need of such prophylactic may be an individual who is genetically predisposed to cancer or at high risk of developing cancer due to various reasons such as a family history of cancer and carcinogenic environment. In one embodiment, the disease may be, for example, joint surgery, bowel surgery, keloid scarring, a disease that produces fibrotic tissue, repetitive motor disorders of a bone tumor.

The proliferative responses associated with organ transplantation that can be treated using this invention include those proliferative responses contributing to potential organ rejections or associated complications. Specifically, these proliferative responses may occur during transplantation of the heart, lung, liver, kidney, and other bodily organs or organ systems. There are provided herein L0XL2 antibodies for use in a method of treating an abnormal proliferative response associated with organ transplantation, comprising administering to a subject in need thereof an effective amount of an anti-L0XL2 antibody; and optionally, a pharmaceutically acceptable carrier or excipient, thereby preventing or reducing abnormal cell proliferation due to organ transplantation. Transplantation may include, for example, transplantation of the heart, lung, liver, kidney, and other bodily organs or organ systems. The indication for the inventive composition also includes fibrosis. Fibrosis results from the abnormal accumulation of fibrous tissue that can occur as a part of the healing process in damaged tissue. Such tissue damage can result from physical injury, inflammation, infection, exposure to toxins, and other causes. Examples of fibrosis include hepatic fibrosis, pulmonary fibrosis, renal fibrosis, cardiac fibrosis, and scleroderma. The compounds and agents of the disclosed invention are also contemplated for the treatment, prevention and / or amelioration of fibrotic conditions.

Fibrotic tissue accumulates in the heart and blood vessels as a result of hypertension, hypertensive heart disease, atherosclerosis, and acute myocardial infarction. Elevated blood pressure, or hypertension, can be caused by a variety of factors and often leads to the development of hypertensive heart disease (HHD) with progression to cardiac arrest and acute myocardial infarction. Likewise, atherosclerosis and other ischemic heart diseases also often result in cardiac arrest. These cardiovascular diseases all exhibit an accumulation of the extracellular matrix or fibrotic deposition which results in the stiffening of the vasculature and stiffening of the cardiac tissue itself. This deposition of fibrotic material is a response to damage induced by the hypertensive and / or sclerotic state, but the effects of this response also result in the negative effects of vascular and cardiac tightening as well as increase of the ventricle. In addition, it is thought that increased cardiac fibrosis observed in cardiovascular disease interrupts or alters the signals transmitted to cardiomyocytes by folding the heart tissue, further leading to disruption of efficient cardiac function and promotion of cardiac arrest and acute myocardial infarction. Given the identified role of increased deposition of the extracellular matrix in cardiac fibroses, the compounds of the present invention are useful in preventing, treating and / or ameliorating cardiac fibroses by inhibiting L0XL2. The present invention also provides compositions, methods, systems, medical devices or kits for treating or preventing cardiac fibrosis associated with cardiovascular diseases, such as hypertensive heart disease (HHD), acute myocardial infarction (MI), atherosclerosis, restenosis (e.g. coronary, carotid and brain lesions) and heart disease associated with ischemic cardiac events. The post-MI healing response may induce L0XL2 expression, but if this process remains to be verified, excessive cross-linking leads to extracellular matrix remodeling or fibrosis resulting in cardiac dysfunction. Enzymes that dismantle matrices and crosslinked collagen or elastin appear to function more slowly or less efficiently and are overcome by crosslinking events. Since L0XL2 also plays a role in the epithelial-mesenchymal transition (EMT), this also contributes to the remodeling of cardiomyocytes and cardiomyocyte hypertrophy, in addition to matrix remodeling.

MI-induced initial repair fibrosis may be useful (eg, prevents aneurysm and related damage) and may be allowed to proceed unimpeded. However, while not wishing to be bound by a particular theory or mechanism of action, the inventors believe that anti-L0XL2 treatment initiated after this stage of repair fibrosis could attenuate reactive (unadaptive) fibrosis leading to cardiac dysfunction. For example, the anti-L0XL2 treatment can be initiated 2, 4, 6, 8, 10, 12, 14, 16, 16, 20, 22, 24, 36 or 48 hours after MI, including all integers therebetween. In addition, anti-LOXL2 treatment can be initiated 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days after MI. Likewise, increases in blood pressure (hypertension) result in increased collagen deposition and reduced protein degradation in cardiac tissue. (Berk et al., J. Clin Invest, 117 (3): 568-575 (2007)). Anti-LOXL2 treatment initiated after diagnosis and / or establishment of hypertensive heart disease or hypertension may prevent, reduce or ameliorate fibrosis associated with hypertension. Such anti-LOXL2 treatment is initiated 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days after elevations of hypertension or systemic blood pressure are diagnosed or detected.

As another example, biomarkers can be used to determine when an appropriate level of cross-linking may be occurring: for example, LOX levels have been shown to correlate with C-reactive protein (CRP), a commonly used biomarker, and the treatment could begin when CRP levels are elevated above normal appropriate levels. More directly, methods and test kits exist to measure the release of crosslinked collagen telopeptides in the urine or blood. Elevated levels of these collagen fragments could indicate a transition from repairing fibrosis to reactive (unadapted) fibrosis. In addition, measurements of cardiac function and yield, including those associated with efficient contraction of the ventricle, may be made.

An L0XL2 inhibitor may be delivered to a subject prior to, concurrently, or following a pathological condition or heart disease, such as hypertension, hypertensive heart disease (HHD), acute myocardial infarction (MI), atherosclerosis, and restenosis, to prevent depletion to reduce the risk of or to regress the pathologic fibrosis associated with such a pathological condition or heart disease. For example, an L0X / L0XL2 inhibitor may be administered at least 1 hour, 2 hours, 3 hours, 5 hours or 10 hours or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, or 14 days after the onset of such pathological condition or heart disease.

In addition, a limited duration of treatment is provided. Treatment should be prolonged only long enough to prevent or attenuate reactive fibrosis to prevent or reduce cardiac dysfunction. For example, short lived FAB antibody fragments are used when short treatment times are desired. Alternatively, whole-length antibodies having a longer serum half-life may be used, with limited dosage over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, even every day between them. Standard cardiac function tests can be used to monitor progress and adjust dosage as needed along with the evaluation of relevant biomarkers discussed earlier. The limited duration of treatment adds to the safety of this approach.

Liver fibrosis is implicated in the pathology of numerous liver diseases. As previously noted, fibrosis occurs as a complication of hemochromatosis, Wilson's disease, alcoholism, schistosomiasis, viral hepatitis, obstruction of the bile duct, exposure to toxins, and metabolic disorders. If unchecked, hepatic fibrosis progresses to cirrhosis (defined by the presence of encapsulated nodules), liver failure, and death. Chronic damage to the liver from such sources as parasites and viral infection (eg HBV, HCV, HIV, schistosomiasis) or long-term stress from alcohol consumption inevitably results in liver remodeling, presumably to encapsulate the damaged area and protect the liver remaining hepatic tissue from the damage. (Li and Friedman, Gastroenterol, Hepatol 14: 618-633, 1999). Hepatic fibrosis results in changes in the extracellular matrix, including increases of 3-10 fold in total collagen content and replacement of the low density matrix matrix with high density, which weaken the metabolic function and synthesis of hepatocytes, Ito cells and endothelial cells. (Girogescu, M., Non-invasive Biochemical Markers of Fibrotic Liver, J. Gastrointestin, Liver Dis., 15 (2): 149-159 (2006)). The compounds of the instant invention are thus useful for the prevention, treatment and / or amelioration of fibrotic liver diseases and such use is contemplated herein by inhibition of LOXL2.

Like hepatic fibrosis, renal fibrosis can result from various diseases and damage to the kidneys. Examples of such diseases and damages include chronic kidney disease, metabolic syndrome, diabetes and resulting glomerular nephritis. It has been recognized that the metabolic syndrome is a group of abnormalities including diabetic distinctions such as insulin resistance as well as central or visceral obesity and hypertension. In almost all cases, glucose deregulation results in stimulation of cytokine release and upregulation of extracellular matrix deposition. Additional factors contributing to chronic kidney disease, diabetes, metabolic syndrome, and glomerular nephritis include hyperlipidemia, hypertension, and proteinuria, all of which result in additional damage to the kidneys and further stimulate extracellular matrix deposition. Thus, irrespective of the primary cause, damage to the kidneys results in renal fibrosis and the concomitant loss of renal function. (Schena, F. and Gesualdo, L., Pathogenic Mechanisms of Diabetic Nephropathy, J. Am. Soc. Nephrol., 16: S30-33 (2005); Whaley-Connell, A., and Sower, JR, Chronic Kidney Disease and the Cardiometabolic Syndrome, J. Clin. Hypert., 8 (8): 546-48 (2006)). The compounds of the instant invention are thus useful for the prevention, treatment and / or amelioration of fibrotic renal diseases (chronic kidney disease, diabetic nephropathy, glomerular nephritis, metabolic syndrome) and such use is contemplated herein. Pulmonary fibrosis includes many syndromes and diseases. Exemplary diseases include idiopathic pulmonary fibrosis (IPF), idiopathic interstitial pneumonia, and adult respiratory distress syndrome (ARDS). The pathogenesis of most pulmonary fibroses, including the above diseases are not well understood, yet they are all characterized by an influx of inflammatory cells and a subsequent increase in the synthesis and deposition of extracellular matrix rich in collagen. (Chua et al., J. Am. Res. Cell Biol., 33: 9-13 (2005); Tzortzaki et al., J. Histochem. & Cytochem., 54 (6): 693-700 2006), Armstrong et al., Am. J. Respir., Crit Care, Med., 160: 1910-1915 (1999)). Given the identified role of collagen deposition and increased extracellular matrix in pulmonary fibroses, the compounds of the present invention are useful for the prevention, treatment and / or amelioration of pulmonary fibroses by inhibition of LOXL2.

Scleroderma is an autoimmune disorder in which there is an overproduction of abnormal collagen. This excess collagen accumulates throughout the body, causing hardening (sclerosis), scarring (fibrosis), and other damage. The damage may affect the appearance of the skin or may involve only the internal organs. The symptoms and severity of scleroderma vary from person to person. Given the identified role of increased collagen in scleroderma, the compounds of the present invention are useful for the prevention, treatment and / or amelioration of scleroderma by an inhibition of L0XL2. Abnormal angiogenesis that can be treated or prevented using this invention includes that abnormal angiogenesis that accompanies rheumatoid arthritis, ischemic reperfusion related edema and brain injury, cortical ischemia, hyperplasia and hypervascularity, (polycystic ovary syndrome), endometriosis, psoriasis, retinopathy diabetic and other angiogenic eye diseases, such as retinopathy of prematurity (retrolental fibroplasia), muscle degeneration, corneal graft rejection, neuroscital glaucoma and Oster Webber syndrome.

Diseases associated with abnormal angiogenesis require or induce vascular growth. For example, corneal angiogenesis involves three phases: a pre-vascular latent period, active neovascularization, and vascular maturation and regression. The identity and mechanism of various angiogenic factors, including elements of the inflammatory response, such as leukocytes, platelets, cytokines and eicosanoids or unidentified plasma constituents are yet to be revealed.

The anti-L0XL2 antibodies described herein may be used to treat diseases associated with abnormal or undesired angiogenesis. The method comprises administering to a patient suffering from abnormal or unwanted angiogenesis a LOXL inhibitor in combination with anti-neoplastic agent or anti-angiogenic agent which is not said LOXL inhibitor. The particular dosage of these agents required to inhibit (partially or completely) angiogenesis and / or angiogenic diseases may depend on the severity of the pathological condition, the route of administration and related factors which may be decided by the attending physician. In general, accepted and effective daily doses are sufficient to effectively inhibit angiogenesis and / or angiogenic diseases.

According to this embodiment, the pharmaceutical formulations of the present invention can be used to treat a variety of diseases associated with undesired angiogenesis, such as retinal / choroidal neovascularization and corneal neovascularization. Examples of retinal / choroidal neovascularization include, but are not limited to, Bests diseases, myopia, optic wells, Stargarts diseases, Pagets disease, venous occlusion, arterial occlusion, sickle cell anemia, sarcoid, syphilis, carotid obstructive diseases of Pseudoxanthoma elasticum, chronic uveitis / vitritis, mycobacterial infections, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, Eales disease, diabetic retinopathy, macular degeneration, Bechets diseases, infections causing retinitis or chroiditis, supposed ocular histoplasmosis, pars planitis, chronic retinal detachment, hyperviscosity syndromes, toxoplasmosis, post-laser trauma and complications, rubella associated diseases (angle neovascularization), and diseases caused by abnormal proliferation of fibrovascular or fibrous tissue including all forms of proliferative vitreoretinopathy . Examples of corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, Vitamin A deficiency, contact lens wear, atopic keratitis, upper limbic keratitis, pterygium keratitis sicca, Sjogrens syndrome, acne rosacea, filectenulose, diabetic retinopathy, reorapathy of prematurity, corneal graft rejection, Mooren's ulcer, marginal degeneration of Terrien, marginal keratolysis, polyarthritis, Wegener's sarcoidosis, scleritis, periphery radial keratotomy, neovascular glaucoma and retrolental fibroplasia, syphilis, Mycobacteria infections, lipid degeneration, burns bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes zoster infections, protozoal infections, and Kaposi's sarcoma.

In yet another embodiment, the pharmaceutical formulations of the present invention may be used to treat chronic inflammatory diseases associated with abnormal angiogenesis. The method comprises administering to a patient suffering from a chronic inflammatory disease associated with abnormal angiogenesis an L0XL2 inhibitor in combination with anti-neoplastic agent or anti-angiogenic agent which is not said L0XL2 inhibitor. Chronic inflammation depends on the continuous formation of capillaries to maintain an influx of inflammatory cells. The influx and presence of the inflammatory cells produces granulomas and thus maintains the state of chronic inflammation. Inhibition of angiogenesis using the compounds described herein may prevent the formation of granulosomes, thereby relieving the disease. Examples of chronic inflammatory disease include, but are not limited to, inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, psoriasis, sarcoidosis and rheumatoid arthritis.

Inflammatory bowel diseases such as Crohn's disease and ulcerative colitis are characterized by chronic inflammation and angiogenesis at various sites in the gastrointestinal tract. For example, Crohn's disease occurs as a chronic transmural inflammatory disease that most commonly affects the distal ileum and colon, but can also occur anywhere in the gastrointestinal tract from the mouth to the anus and perianal area. Patients with Crohn's disease generally have chronic diarrhea associated with abdominal pain, fever, anorexia, weight loss, and abdominal bloating. Ulcerative colitis is also a chronic, nonspecific, inflammatory and ulcerative disease that arises in the colonic mucosa and is characterized by the presence of bloody diarrhea. These inflammatory bowel diseases are usually caused by chronic granulomatous inflammation along the gastrointestinal tract, involving new capillary outbreaks surrounded by a cylinder of inflammatory cells. Inhibition of angiogenesis by the pharmaceutical formulations of the present invention should inhibit the formation of outbreaks and prevent the formation of granulomas. Inflammatory bowel diseases also exhibit extra intestinal manifestations, such as skin lesions. Such lesions are characterized by inflammation and angiogenesis and may occur at many sites beyond the gastrointestinal tract. Inhibition of angiogenesis by the pharmaceutical formulations of the present invention should reduce the influx of inflammatory cells and prevent the formation of lesions. Therefore, L0XL2 antibodies are provided herein for use in a method of treating an inflammatory bowel disease, comprising administering to a subject in need thereof an effective amount of an anti-L0XL2 antibody; and optionally, a pharmaceutically acceptable carrier or excipient.

Sarcoidosis, another chronic inflammatory disease, is characterized as a multi-system granulomatous disorder. Granulomas of this disease can form on any side of the body and the symptoms thus depend on the location of the granulomas and whether the disease is active. Granulomas are created by angiogenic capillary outbreaks that provide a constant supply of inflammatory cells. In using the pharmaceutical formulations of the present invention to inhibit angiogenesis, such formation of granulomas can be inhibited. Psoriasis, also a recurrent and chronic inflammatory disease, is characterized by papules and plaques of various sizes. Treatment using the pharmaceutical formulations of the present invention should prevent the formation of new blood vessels necessary to maintain the characteristic lesions and provide the patient with relief of symptoms. Therefore, there is provided herein a method of preventing the formation of new blood vessels necessary to maintain the characteristic lesions and provide the patient with relief of symptoms, comprising administering to a subject in need thereof an effective amount of an anti-L0XL2 antibody; and optionally, a pharmaceutically acceptable carrier or excipient. Rheumatoid arthritis (RA) is also a chronic inflammatory disease characterized by nonspecific inflammation of the peripheral joints. The blood vessels in the sinuvial lining of the joints are thought to undergo angiogenesis. In addition to forming new vascular networks, endothelial cells release reactive oxygen species and factors that lead to tissue growth and cartilage destruction. Factors involved in angiogenesis can actively contribute to, and help maintain, the chronically inflamed state of rheumatoid arthritis. Treatment using the pharmaceutical formulations of the present invention alone or in conjunction with other anti-RA agents may prevent the formation of new blood vessels necessary to maintain chronic inflammation and provide the patient with RA symptom relief. Other anti-RA agents are conventional and known in the art. Therefore, L0XL2 antibodies are provided herein for use in a method of preventing or treating RA, comprising administering to a subject in need thereof an effective amount of an anti-LOX antibody or an anti-L0XL2 antibody; and optionally, one or more other anti-RA agents.

In addition to the use of the anti-LOXL2 antibodies alone in the treatment of the indications described above, combination therapy is also contemplated herein. The methods provided herein may further include administering an anti-cancer agent or treatment to the patient. There is provided herein a method of treating any of the indications described above by administering an anti-LOX antibody and an anti-L0XL2 antibody.

In one aspect, this invention contains methods for inhibiting invasion and metastasis of tumor cells by contacting the cells with at least one cytotoxic agent and at least one anti-L0X2 antibody. In general, the method includes a step of contacting metastatic tumor cells with an amount of at least one cytotoxic agent and at least one anti-L0X2 antibody, which, in combination, is effective to reduce or inhibit the invasion or metastatic potential of the cell . Alternatively, according to the present invention, an anti-L0XL2 antibody can be combined with a chemotherapeutic agent to sensitize tumor cells (e.g., transition from the EMT state to the MET state) to be killed by the chemotherapeutic agent, thus not only preventing or inhibiting tumor invasion and metastasis, but also inhibiting primary tumor growth.

Any suitable anti-cancer agent may also be employed in the present methods.

As used herein the term &quot; chemotherapeutic agent &quot; or &quot; chemotherapeutic &quot; (or &quot; chemotherapeutic, &quot; in the case of treatment with a chemotherapeutic agent) is intended to encompass any non-proteinaceous (i.e. non-peptidic) chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide (CYTOXAN ™); alkyl sulfonates, such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethylenimines and methylamelamines including altretamine, triethylenehemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bulatacin and bulatacinone); a camptothecin (including synthetic analogue topotecan); bryostatin; calistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptoficins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CBI-TMI); eleuterobina; pancratistatin; a sarcodictine; spongistatin; nitrogen mustards such as chlorambucil, clomafazine, colophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembiquin, fenesterin, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocine, foramustine, lomustine, nimustine, ranimustine; antibiotics such as enedine antibiotics (for example calicheamicin, especially calicheamicin gamma II and calicheamicin phill, see, for example, Agnew, Chem. Inti, Ed. Engl., 33: 183-186 (1994); , bisphosphonates, such as clodronate, a esperamicin, as well as chromophore neocarzinostatin and chromophores antibiotics related chromoprotein enedine), actinomycin, actinomycin, autramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinoophyline, chromomycins, dactinomycin, daunorubicin, detorubicin, (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrroline-doxorubicin and deoxidoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid , nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozoc ina, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as demopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprin, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenishers such as frolinic acid; aceglatone; aldofosfamide glycoside; aminolevulinic acid; enyluracil; amsacrine; bestrabucilo; bisantrene; edatraxate; defofamina; demecolcin; diaziquone; elfornitine; eliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinane; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; fenameto; pyarubicin; losoxantrone; podophylinic acid; 2-ethylhydrazide; procarbazine; PSK ™; reasonable; rhizoxin; sizofiran; spirogermânio; tenuazonic acid; triaziquone; 2,2 ', 2 "-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethane; vindesine; dacarbazine; manomustine; mitobronitol; mitolactol; pipobroman; gacitosine; arabinoside (&quot; Ara-C &quot;);cyclophosphamide;thiopeta; taxoids, for example, paclitaxel (TAXOL ™, Bristol Meyers Squibb Oncology, Princeton, N.J.) and docetaxel (TAXOTERE ™, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (Gemzar ™); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitroxantrone; vancristine; vinorelbine (Navelbine ™); novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeoloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Also included in the definition of &quot; chemotherapeutic agent &quot; anti-hormonal agents which act to regulate or inhibit hormone action in tumors such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including Nolvadex ™), raloxifene, droloxifene, 4-hydroxytamoxifen , trioxifene, ceoxifen, LY117018, onapristone and toremifene (Fareston ™); (5) -imidazoles, aminoglutethimide, megestrol acetate (Megace ™), exemestane, formestane, fadrozole, vorozol (Rivisor ™), and the like, which inhibits the production of estrogen in the adrenal glands, such as, for example, letrozole (Femara ™) and anastrozole (Arimidax ™); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

In a non-limiting example, this invention includes methods for synergistically inhibiting the invasion and metastasis of tumor cells by contacting cells with at least cisplatin and at least one anti-LOX antibody (see Figure 18). One who practices the methods described herein should understand that an anti-LOX2 antibody could be used in such methods.

In one embodiment, the anti-neoplastic agent in combination with the LOX / LOXL modulator is a tyrosine kinase inhibitor. For example, ZD1839 (Iressa ™ from AstraZeneca K.K.) shows a competitive effect for ATP at the EGFR (epidermal growth factor receptor) ATP binding site (tyrosine kinase receptor) and inhibits tyrosine kinase activity by inhibiting tyrosine kinase autophosphorylation. As a result, the anticancer effect is expressed by blocking a transduction of EGFR-encoding signal (ligands such as epidermal growth factor (EGF) are linked to the extracellular domain of EGFR, followed by activation of EGFR tyrosine kinase in the intracellular domain, causing not only EGFR autophosphorylation but also the phosphorylation of various intracellular target proteins, then transducing a cell surface proliferation signal to the nucleus, then transducing the proliferation signals from the surface of the cancer cell to the nucleus and resulting in the proliferation, infiltration, metastasis, angiogenesis of cancer cells) in association with proliferation, infiltration, differentiation and metastasis. IMC-C225 or cetuximab (Erbitux ™) which is an EGFR-directed monoclonal antibody) recognizes the EGFR receptor part on a cell membrane surface and inhibits EGFR autophosphorylation thereby inhibiting tyrosine kinase activity. Herceptin is a monoclonal antibody against Her2 / Neu that is homologous to EGFR and imatinib mesylate (GLEEVEC ™, formerly STI-571) can inhibit both BCR-Abl and c-kit tyrosine kinase activities (non-patent document No. 2 ). Sorafenib (Nexavar ™) is a small molecular inhibitor of Raf kinase, PDGF (platelet-derived growth factor), VEGF receptor 2 &amp; 3 kinases and c-kit.

As used herein, monoclonal antibodies against antigen tumors are antibodies triggered against antigens expressed by tumors and leukemic cells, preferably tumor specific antigens. The monoclonal antibody also includes humanized and fully human antibody.

Other examples of therapeutic antibodies for cancer therapy include Trastuzumab (HERCEPTIN ™; overexpression of HER2 protein is associated with more aggressive disease and weaker prognosis in the clinic); Rituximab (RITUXAN ™) that is raised against CD20 in lymphoma cells and selectively depletes pre-B and B mature normal and malignant CD20 + cells; Alemtuzumab (CAMPATH ™), a monoclonal antibody that specifically targets the CD52 antigen that is found on B and T lymphocytes and used for the treatment of chronic lymphocytic leukemia (CLL) and lymphoma; and Gemtuzumab zogamicin (MYLOTARG ™), an antibody conjugate that combines a CD33-specific antibody with a chemotherapeutic drug (zogamycin) and is indicated for the treatment of recurrent adult acute myelocytic leukemia.

In another embodiment, the anti-angiogenic agent is combined with an L0XL2 inhibitor to treat cancer and other diseases associated with abnormal or undesirable angiogenesis. Examples of anti-angiogenic agents include, but are not limited to, retinoid acid and derivatives thereof, 2-methoxyestradiol, ANGIOSTATIN ™, ENDOSTATIN ™, suramin, squalamine, metalloproteinase I tissue inhibitor, metalloproteinase-2 tissue inhibitor , plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, cartilage-derived inhibitor, paclitaxel, platelet factor 4, protamine sulfate (clupeine), sulfated chitin derivatives (prepared from queen crab shells) (sp-pg) peptidoglycan complex, staurosporine, matrix metabolism modulators, including, for example, proline analogs ((I-azetidine-2-carboxylic acid (LACA), cishydroxyproline, d, 1-3, 4-propyl-5- (4-pyridinyl) -2 (3 H) -oxazolone, methotrexate, mitoxantrone, heparin, interferons, 2-macroglobulin-serum, chimp-4-dehydroproline, thiaproline, α-dipyridyl, β-aminopropionitrile fumarate, 3, chemostatin, tetradecassu β-cyclodextrin acetate, eponemycin; fumagillin, sodium thiomalate gold, d-penicillamine (CDPT), beta.-1-anticholagenase-serum, alpha.2-antiplasmin, bisanthrene, disodium lobenzarite, disodium n-2-carboxyphenyl-4-chloroanthronyl acid, or "CCA &quot; , thalidomide; angiostatic steroid, carboxynnaminolmidazole; metalloproteinase inhibitors, such as BB94. Other anti-angiogenesis agents include antibodies, preferably monoclonal antibodies against these angiogenic growth factors: bFGF, aFGF, FGF-5, VEGF, VEGF-C, HGF / SF and Ang-1 / Ang-2 isoforms. Ferrara N. and Alitalo, K. &quot; Clinical application of angiogenic growth factors and their inhibitors &quot; (1999) Nature Medicine 5: 1359-1364.

Exemplary anti-fibrotic agents include, but are not limited to, compounds such as β-aminoproprionitrile (BAPN), as well as the compounds disclosed in U.S. Patent No. 4,965,288 to Palfreyman, et al., Issued October 23, 1990, entitled &quot; Inhibitors of lysyl oxidase, relating to inhibitors of lysyl oxidase and their use in the treatment of diseases and conditions associated with the abnormal deposition of collagen; U.S. Patent No. 4,997,854 to Kagan, et al. , issued March 5, 1991, entitled &quot; Anti-fibrotic agents and methods for inhibiting the activity of lysyl oxidase in situ using adjacently positioned diamine analogue substrate, &quot; in relation to compounds that inhibit LOX for the treatment of various pathological fibrotic conditions. Additional exemplary inhibitors are described in U.S. Patent No. 4,943,593 to Palfreyman, et al. , issued July 24, 1990, entitled &quot; Inhibitors of lysyl oxidase, &quot; with respect to compounds such as 2-isobutyl-3-fluoro-, chloro-, or bromo-allylamine; as well as, for example, U.S. Patent No. 5,021,456; U.S. Patent No. 5,505,917; U.S. Patent No. 5,120,764; U.S. Patent No. 5,181,297; U.S. Patent No. 5,252,608 (to 2- (1-naphthyloxymethyl) -3-fluoroallylamine). Exemplary anti-fibrotic agents also include the primary amines which react with the carbonyl group at the active site of the lysyl oxidases, and more particularly those which produce, upon attachment to the carbonyl, a resonance-stabilized product such as the following primary amines: ethylenediamine , hydrazine, phenylhydrazine and its derivatives, semicarbazide and urea derivatives, aminonitriles such as beta-aminopropionitrile (BAPN) or 2-nitroethylamine, unsaturated or saturated haloamines such as 2-bromoethylamine, 2-chloroethylamine, 2-trifluoroethylamine, 3- bromopropylamine, p-halobenzylamines, selenohomocysteine lactone. In another embodiment, the anti-fibrotic agents are copper chelating agents, penetrating the cells or not. Additional exemplary compounds include indirect inhibitors such as compounds which block the aldehyde derivatives that originate from oxidative deamination of lysyl and hydroxysilane residues by lysyl oxidases, such as thiolamines, in particular D-penicillamine or its analogues such as 2-amino- D-2-amino-3-methyl-3 - ((2-acetamidoethyl) dithio) butanoic acid, p-2-amino-3-methyl-3 - ((2-aminoethyl) dithio) butanoic acid, sodium sulfinate 4 - ((p-1-dimethyl-2-amino-2-carboxyethyl) dithio) butane, 2-acetamidoethyl-2-acetamidoethanethiol sulfanate, sodium trihydrate 4-mercaptobutanesulfinate.

The present methods can be performed on cells in culture, for example in vitro or ex vivo, or can be performed on cells present in a subject, for example, as part of an in vivo therapeutic protocol. The therapeutic regimen may be performed on a human subject or other animals. The anti-LOX2 antibody provided herein may be administered in any order relative to the chemotherapeutic agent. Sometimes, the anti-LOX2 antibody and the agent are administered simultaneously or sequentially. They can be given in different places and at different dosing regimens. The enhanced therapeutic efficacy of the combination therapy of the present invention represents a promising alternative to the highly toxic conventional regimens of anticancer agents. Chemotherapeutic agents to be employed in such methods have been described in more detail above. EXAMPLES EXAMPLE 1 Methods of generating Anti-LOX and Anti-LOXL2 Murine Monoclonal Antibodies

Mice (BALB / c (00467)) were injected subcutaneously (s.c.), 5 times at 2-3 week intervals with 0.05 mg antigen (Ag) in an adjuvant formulation. For Ags peptides, the peptides were conjugated to bovine serum albumin and formulated in Freunds Adjuvant (FA) prior to immunization. For Ags proteins, the protein was formulated in Alhydrogel-Muramyl dipeptide adjuvant (ALD / MDP).

Mice were injected with Ag formulated in PBS, each day for 3 days through a combination of s.c., intraperitoneal (i.p.) and intravenous (i.v.) routes, 0.05 to 0.1 mg / lane. Spleen cells and lymph nodes of the mice were isolated and fused with P3X63-Ag8.653 myeloma cells using 50% polyethylene glycol. Cells were cultured and a hybridoma library of selected HAT cells was isolated essentially as described in Kenney, et al. (&quot; Production of monoclonal antibodies using a secretion capture web report. &quot; Biotechnology 13: 787-790, 1995). The hybridoma library was cloned using a fluorescent activated cell sorter with automatic cell deposition unit. Single viable cells were classified into 96-well plates based on the forward dispersion, sidestream and fluorescence criteria of propidium iodide as described by Kenney et al.

Sera and supernatants were sorted by enzyme-linked immunosorbent assay (ELISA) using Ag-coated microtiter plate wells, which were then incubated with mouse plasma or hybridoma supernatant, followed by antibody-HRP IgG conjugate (Fc-specific) of goat mouse followed by the TMB substrate solution and termination reagent.

The plate wells were washed to remove unbound antibody or antigen between all incubations and the results were determined.

The VH and VL amino acid sequences of an anti-L0XL2 murine monoclonal antibody identified using the described methods are provided in Figure 6A and Figure 6B, respectively. For each variable region, the signal peptides are shown in italics, the CDRs are underlined and the beginning of the constant structure is shown in bold.

A V H amino acid sequence of an anti-LOX murine monoclonal antibody identified using the methods described is provided in Figure 7A. Two VL amino acid sequences of anti-LOX murine monoclonal antibodies identified using the methods described are provided in Figures 7B and 7C. For each variable region, the signal peptides are shown in italics, the CDRs are underlined. EXAMPLE 2

Anti-L0XL2 antibodies were classified using a protein B screening update to evaluate the enzymatic activity of L0XL2.

Antibody candidates were initially chosen based on ELISA point tests. ELISA was performed on multiple antigens by Antibody Solutions and antibodies showing strong ELISA signal on the antigen of interest were selected for further characterization in enzymatic assays. L0XL2 produces hydrogen peroxide when the 1,5-diaminopentane substrate is deaminated and the enzyme regenerated.

Antibodies were assessed for their ability to inhibit enzymatic activity using a biochemical assay that binds peroxide production (released by L0XL2) to HRP and measuring the conversion of red amplex into fluorescent product. The antibody hybridoma supernatant (10 æl) was added to 40 æl enzyme mixture (62.5 mM sodium borate pH 8.0, 5 units / ml HRP, 125 nM LOXL2, 10 ppm antifoam) and incubated at at room temperature for 1 hour on a 96-well plate with completely black area. The enzymatic reaction was started by the addition of 50 æl of substrate solution (50 mM sodium borate, 100 mM amplex red reagent, 20 mM 1,5-diaminopentane (DAP), 10 ppm antifoam) and read in a reader. Molecular Devices M5 plate at 37 ° C. The plate reader was set to read fluorescence (ex = 544 nm, at = 590 nm) in kinetic mode for 1 hour. Data were recorded as the slope of the fluorescence response over time. These slopes were compared to a control in which the hybridoma media were added to the enzyme mixture. The smaller slopes of the control were considered inhibitors.

The M1 (asc), M4, M1, M13, M12, M16, M19, M20, M20, M20 (asc) antibodies and M25 test antibodies were tested against BAPN (a competitive inhibitor of LOXL2) as a positive control eLOXL2 as a control negative (see Figure 8).

An anti-LOXL2 antibody was designated AB0023. Anti-LOXL2 antibodies repeated the inhibitory activity observed in 10 ml of preparation materials in the enzyme assay. Inhibition was also repeated in cell-based assays (see below). Sequence analysis confirmed that the amino acid sequences of M01, M16, M19 and M20 are identical. EXAMPLE 3

Anti-LOXL2 antibody AB0023 and enzymatic activity The enzymatic activity of the anti-L0XL2 antibodies can be evaluated and the IC50s determined.

M1, M1 (asc), M20 and M20 (asc) were evaluated in the presence of 25 nM L0XL2 and 15 mM 1.5 DAP on increasing concentrations of antibody.

Determinations of IC 50

Dose responses in selected antibodies were performed against L0XL2 using the enzyme-linked assay described above. A series of antibody dilutions were made in PBST (0.01% tween-20) and 10 Âμl of this was added to 40 Âμl of the enzyme mixture (62.5 mM sodium borate pH 8.0, 5 units / ml HRP , 125 nM LOXL2, 10 ppm antifoam) and incubated at room temperature for 1 hour in a 96-well fully blacked-out plate. The enzymatic reaction was started with the addition of 50 æl of substrate solution (50 mM sodium borate, 100 mM amplex red reagent, 20 mM 1,5-diaminopentane, 10 ppm antifoam) and read in an M5 plate reader using conditions described above. The slopes of the fluorescence response as a function of time were plotted against the antibody concentration and the data adjusted to a four-parameter adjustment using GraFit. The center point of this graph is the apparent IC50 and is the concentration at which fifty percent of the total response is inhibited.

Ab0023 has been shown to be a partial inhibitor of the enzymatic activity of LOXL2 with an apparent IC 50 of approximately 30 nM (see Figure 9).

Based on the use of a partial inhibitor in the clinic for therapeutic treatment (i.e., Nevirapine - according to the approved HIV-1 drug described by Spence et al. (1995) Science 267), a partial inhibitor of L0XL2 can also be used in therapeutic applications . EXAMPLE 4

Anti-LOXL2 antibody AB0023 is a noncompetitive inhibitor The activity of the anti-L0XL2 antibody AB0023 was evaluated on increasing concentrations of 1.5 DAP and on increasing concentrations of antibody (1 μ, 0.005 μ, 0.050 μ and 0.300 μ).

Mode of inhibition The mode of inhibition of antibodies against LOXL2 was conducted using the model described below. In these experiments, the steady state rate dependence of the 1,5-diaminopentane concentration was monitored at increasing concentrations of the antibody. The objective was to evaluate if the Km for the substrate, kcat or both alter in the presence of antibody. The data collected were analyzed globally with Grafit using the model shown in the following figure. E represents enzyme, S represents substrate, A represents antibody and P represents product. The α parameter describes the effect of the compound on substrate affinity. An α value equal to one describes a situation in which the compound binds equally well to the free enzyme and the enzyme-substrate complex (noncompetitive inhibition type). Values less than one describe an interaction in which the compound binds the enzyme-substrate complex (type-inhibiting type). Values greater than one correspond to the binding of the compound to the free enzyme better than to the enzyme-substrate complex (competitive inhibition type). Β value describes the effect of the modulator on the rate of the enzyme. Inhibitors have values less than one (for a complete inhibitor β = 0) and the activators have values greater than one. KA is the dissociation constant of the compound, Ks is the Michaelis constant for the substrate and k is the catalytic rate of the enzyme. Steady state rates were determined from the slope of the fluorescence response as a function of time, as previously described (determination of IC 50). The data were plotted as the steady-state concentration dependence of the substrate concentration (1,5-diaminopentane) at various fixed antibody concentrations and analyzed with GraFit.

Anti-L0XL2 antibody AB0023 was determined to be a noncompetitive inhibitor based on the following results: a = 1, K ± 0.067 and β = 0.5 (see Figure 10). EXAMPLE 5

Kinetic measurement of antibody ΆΒ0023 binding to LOXL2 by surface plasma resonance The binding affinity and dissociation rate of AB0023 were evaluated by surface plasma resonance (SPR).

Binding affinities were measured using a Bio-Rad ProteOn instrument with thermostat at 25 ° C. Binding affinities were determined using two methods, using amine coupling; one in which the antibody was immobilized and the antigen (LOXL2) was added and another in which the antigen (LOXL2) was immobilized and the antibody was added. The antibody or antigen was immobilized on a GLC chip using a ratio of 1: 1 NHS to EDC provided with the ProteOn titration kit. The chip was first activated with an NHS / EDC mixture and then the antigen or antibody at 1 pg / ml in acetate buffer pH 4.5 was eluted on the activated surface to be bound. This typically produces a coupling of about 500 RU's. The activated surface of the chip was then capped with the addition of 1M ethanolamine. The bound chips were stored at 4 ° C and regenerated with 50 mM sodium hydroxide.

Dissociation constants were determined by probing the coupled chip with a series of antibody or antigen dilutions in PBST (0.05% Tween-20). The attachments were acquired on all six channels available on the ProteOn using a non-coupled channel as a reference. The data collected were analyzed using Bio-Rad's ProteOn management program.

It has been found that AB0023 binds closely to LOXL2 and is released slowly. Kd was estimated to be 0.1 -1.0 nM. In addition, Î "AB0023 has been found to have the following characteristics: kon = 1.68 x 106 M_1s_1, koff = 1.17 x 10 -4s -1, K D = 0.69 nM and ti / 2 = 98.7 minutes. To see

Figure 11. The mapping of the domain was conducted and it was found that AB0023 binds to the SRCR3-4 domain.

Materials and methods

All plates were obtained from Corning. The secondary antibody and the Pico substrate were obtained from Pierce. The strong root peroxidase (HRP) was obtained from Sigma. All ProteOn reagents were obtained from Bio-Rad. LOXL2 was obtained from R & D Systems. Antibodies used in this study are produced in an Antibody Solution or through ascites from Aragen Biosciences. All other reagents were of the highest possible quality.

ELISA Binding Antibody binding to LOXL2 was determined using a Luminescence-based ELISA. Plates

Corning were coated with 0.1 pg / ml of LOXL2 or antigen of interest in 50 mM borate buffer (pH 8.0) overnight at 4 ° C. The plates were washed using BioTek plate washer and blocked with 5% lean milk in PBST (0.05% tween-20) for 1 hour at room temperature. Plates were washed with PBST (0.05% tween-20) and then used immediately or stored at 4 ° C in a desiccator for future use. The antibody body to be tested was serially diluted in PBST (0.01% tween-20) and 100 μl of each dilution was added per well. Plates were incubated with test article for 1 hour at room temperature and then washed with PBST (0.05% Tween-20). Detection antibody (anti-mouse HRP conjugate) was diluted 16,000 fold in 5% lean milk in PBST (0.05% Tween-20) and 100 Âμl were applied per well. The plates were incubated for 1 hour with the detection antibody and then washed with PBST (0.05% PBST). The signal was detected using Pierce's SuperSignal peak ELISA chemiluminescent substrate according to the manufacturer's instructions. Luminescence was measured using a Molecular Devices M5 plate reader with an integration time of 500 ms which captures all wavelengths. The data were background corrected and the antibody concentration luminescence signal dependence was adjusted using the Langmuir isothermal equation using the GraFit program. At times when the antigen concentration was similar to the dissociation constant, the quadratic tight binding equation was used. The reported dissociation values were obtained from the adjustments to these equations; where PL represents the signal of the bound complex, B max is that maximum binding, K D is the dissociation constant and L is the concentration of the ligand.

Isothermal equation of Langmuir:

Tight bonding equation:

The AB 0 02 3 was tested against MCD-LOXL2, LOXL2 (R &amp; D), SRCR1-2 and SRCR 1-4. It has been found that AB0023 binds to the SRCR3-4 domain (see Figure 12). EXAMPLE 7

Inhibition of migration / invasion in Collagen I and Collagen IV and inhibition of cell growth

Cell-based assays were conducted to evaluate binding of AB0023 to bind substrate (i.e., collagen).

In short, ο AB0023 was amplified from several samples before the test.

Invasion kits from collagen I and Cultrex 96 well IV collagens (Trevigen, Gaithersburg, MD) were used for classifications of antisera / antibody supernatants. MDA MB cells were deprived of serum 24 hours before the assay was assembled. On the day of the assay, plates coated with collagen I and collagen IV were made at least 4 hours before the invasion assay was assembled (no more than 8 hours earlier). Plasmas with collagen I and collagen IV were coated according to the manufacturer's instructions. The cells were plated at 20,000 cells per well in 95æl of serum free media in the upper chamber of the plate. One hundred and fifty (150) of media containing 10% FBS and 1x L-glutamine were aliquoted into the lower chambers of the plate. Using a multichannel pipette, 5 pcs of each antiserum was placed in the upper chambers of the plate. The antiserum and cell mixture was carefully mixed up and down once with the pipette. The plates were incubated at 37øC with 5% CO2 for 48 hours.

After 48 hours the plates were ready to be read. The cellular dissociation solution containing calcein AM was made, according to the manufacturer's instructions. The plates were also washed and disassembled according to the manufacturer's instructions. 125 pcs of cellular dissociation solution containing calcein AM was added to the wells of the lower chambers and the plates were placed at 37øC for 30 minutes. After 30 minutes the sides of the plates were lightly touched to loosen the cells and the plates were placed in an incubator for an additional 30 minutes at 37 ° C. The plates were then disassembled and the bottom plate was placed on a plate reader (SpectraMax M5, Molecular Devices, Sunnyvale, Ca) with the definitions: Fluorescence, emission 485-520, top reading, black opaque plate, sensitivity to 30. A inhibition of migration / consistent invasion was observed in collagen I (Figure 13) and collagen IV, from the suppressants through 10 ml preparation material and expanded to 100 ml preparations and ascites.

Cell adhesion assays MDA-MB231 cells were plated with 15 cm 2 and grown in 4.5 g / L glucose containing DMEM (10% FBS and 2 mM L-Glutamine) to be confluent on the day of the assay. The media were aspirated and the cells were washed 2 times with 10 ml 1 mM EDTA PBS per dish. The fram cells are removed from the plates by incubating with another 10 ml 1 mM EDTA PBS for 5 minutes at 37 ° C in a biosafety cabinet and subsequently pipetting the cells to the plate in a PBS EDTA solution. Cell concentration was determined and cells sufficient for the assay (50k / well plus pipetting) were spun into 15 ml conical tubes. The cell pellet was dispersed in serum-free DMEM prewarmed to 500K cells / ml and CuCl 2 was added to a final concentration of 1 μΜ. One hundred (100) μΐ / cell suspension well was pipetted into a U-shaped bottom 96-well tissue culture dish containing 10 μΐ of appropriate mAb dilution. The cell suspension / mAb mixture was allowed to incubate for 10 minutes at room temperature in the dark. One hundred (100) μΐ / well of the resuspended cell / mAb mixture was then transferred to 96 well plates coated with collagen IV (BD Biocoat, BD Biosciences). The plates were incubated at 37 ° C in a biosafety cabinet for 1 hour. The wells were then aspirated and gently washed twice with 200 μΐ DPBS (Mediatech) to remove unbound cells. One hundred (100) μΐ final concentration of 10 μM Calcein-AM (BD Biosciences) in DPBS was then added to each well to stain remaining cells. The plates were incubated at 37 ° C in a biosafety cabinet for 1 hour. Plates were read on a Molecular Devices M5 plate reader at 494/517 (excitation / emission). The percent (%) of adhesion was calculated by normalizing to unrelated PBS or antibody controls.

Using this assay, partial inhibition of adhesion was observed at the time of exposure of the cells to antibody AB0023.

Cell growth

Anti-LOXL2 antibodies inhibited cell growth of four cell lines: 231 is a breast cancer cell line, BT549 is a breast cancer cell line, HT1080 is a fibrosarcoma and BxPC3 is a line of prostate cancer cells (Figure 17 ). Thus, the antibody is effective in inhibiting the growth of cancers of different origins. EXAMPLE 8

Inhibition of ΆΒ0023 of EMT type change

Epithelial to mesenchymal changes were evaluated using immunohistochemistry.

To detect whether a cell is in an EMT or mesenchymal-to-epithelial (MET) transition state, the cells were stained with specific cellular protein markers for epithelial or mesenchymal states such as E-cadherin, vimentin, fibronectin, and phalloidin for detect F-actin.

Rhodamine staining protocol phalloidin

The cells were seeded 24 hours before the day of staining; the cells were approximately 80% confluent 24 hours later on an 8 chamber slide. The next day the media were aspirated and the chambers were rinsed with IX PBS. The cells were then fixed with 4%

Paraformaldehyde (PFA) for 20 minutes at room temperature and then rinsed once with IX phosphate buffered saline (PBS). For permeabilization, cells were treated with 0.5% Saponin (JT Baker,

Phillipsburg, NJ) in PBS for 5 minutes at room temperature. The chambers were carefully rinsed once with IX PBS and a 1: 100 dilution of rhodamine phalloidin (Invitrogen, Carlsbad, Ca) in PBS was added to the cells and incubated for 15 minutes at room temperature. The chambers were rinsed twice with IX PBS and the blades were assembled with Vectashield (Vector Laboratories, Burlingame, CA).

E-cadherin staining protocol

The cells were seeded 24 hours before the day of staining; the cells were approximately 80% confluent the next day on an 8 chamber slide. The next day the media were aspirated and the chambers were rinsed with IX PBS. The cells were then fixed with ice cold methanol and then incubated for 2 minutes at -20 ° C. Cells were rinsed once with IX PBX and 1æg / ml E-cadherin Ab (Calbiochem, Gibbstown, NJ) was added to the slide chambers. The slides were then incubated at 37 ° C for 1 hour. After thoroughly rinsing the chambers once with IX PBS, the secondary Ab (anti-mouse IgG cy3 conjugate, Jackson Immuno Research, West Grove, Pa) was added and incubated at room temperature for 30-45 minutes. The chambers were rinsed twice with IX PBS and mounted with Vectashield (Vector Laboratories, Burlingame, CA).

Means of HS-578t cells (high L0XL2) applied to MCF-7 cells (low L0XL2 / negative). Cells were stained with rhodamine-phalloidin (F-actin, red) and Dapi (nuclei, blue).

AB0023 has been shown to inhibit EMT-like changes induced by the conditioned media of tumor cells expressing L0XL2 (data not shown). EXAMPLE 9

Anti-L0XL2 antibody AB0023 binds the matrix-associated L0XL2.

Ab internalization and uptake studies in Hs578t cells.

Hs578t cells were cultured in DMEM containing 10% FBS and 1x glutamine. The cells were seeded on an 8-chamber glass slide (BD Falcon, Franklin Lakes, NJ) and allowed to adhere overnight. For low confluence, the cells were seeded at 30-40,000 cells per slide. Low confluency was used for detection of Lox in the cytosol 24 hours later. For high confluence, the cells were seeded at 100,000 cells per slide. High confluency was used for detection of Lox associated with matrix and collagen approximately 48-72 hours later.

The next day, 1 pg / ml (final concentration in the regular growth medium) of anti-Lox M64 monoclonal Ab or anti-Loxl2 mAb (mAb) was added to the chambers. For continuous uptake, the mAbs were incubated with cells at different time points: for example, 3 hours, 8 hours or 24 hours (overnight). After appropriate amount of uptake is continued, the media were removed and the chambers were rinsed with IX PBS. Cells were fixed in 4% PFA (paraformaldehyde) at room temperature for 20 minutes. After fixation, cells were washed with IX PBS at room temperature for 5 minutes and then quenched in 50 mM ammonium chloride at room temperature for 10 minutes. Cells were washed again with IX PBS at room temperature for 5 minutes.

Cells were permeabilized by adding saponin buffer (0.5% Saponin / 1% BSA in PBS) at room temperature for 20 minutes. Secondary detection Ab (Alexa Fluor 488 anti-mouse IgG, Invitrogen, Carlsbad, CA) was added at room temperature in saponin buffer and the cells were incubated for 30-45 minutes. The cells were then washed 3X in saponin buffer. The slides were mounted with vectashield (Vector Laboratories, Burlingame, CA).

For detection of collagen detection, the cells were incubated with anti-collagen antibody (1:50, Calbiochem anti-collagen type I polyclonal rabbit, Gibbstown, NJ) one hour prior to setting the cells with 4% PFA. The secondary Ab for collagen used is Cy3 from rabbit anti-rabbit (ImmunoJackson Labs, West Grove, PA). Immunoblot and immunofluorescence analysis (data not shown) indicated that LOXL2 was predominantly intracellular and low density but secreted at high cell density (confluent cells). LOXL2 was detected in the confluent cell media and also in the extracellular matrix. Immunofluorescence in living cells indicated that AB0023 bound LOXL2 associated with the collagen matrix. EXAMPLE 10

Anti-LOX M64 Antibody LOX The activity of the anti-LOX M64 antibody was assessed on increasing concentrations of 1.5 DAP and on increasing concentrations of antibody (see Figure 15). Materials and methods

All plates were obtained from Corning. The secondary antibody and Pico substrate were from Pierce. Amplex red reagent was from Invitrogen. The strong-root peroxidase (HRP), 1,5-diaminopentane, antifoam were from Sigma. All ProteOn reagents were from Bio-Rad. LOX was produced internally in Arresto Biosciences. The antibodies used in this study were produced in Antibody solution or through ascites (ase) from Aragen Biosciences. All other reagents were of the highest possible quality.

ELISA Binding Antibody binding to LOX was determined using a luminescence-based ELISA. White Corning plates were coated with 0.1æg / ml LOX or antigen of interest in 50mM borate buffer (pH 8.0) overnight at 4 ° C. Plates were washed using a BioTek plate washer and blocked with 5% lean milk in PBST (0.05% tween-20) for 1 hour at room temperature. Plates were washed with PBST (0.05% tween-20) and then used immediately or stored at 4 ° C in a desiccator for future use. The antibody to be tested was serially diluted in PBST (0.01% tween-20) and 100æl of each dilution was added per well. The plates were incubated with test material for 1 hour at room temperature and then washed with PBST (0.05% tween-20). Detection antibody (anti-mouse HRP conjugate) was diluted 16,000 fold in 5% lean milk in PBST (0.05% tween-20) and 100æl were applied per well. The plates were incubated for 1 hour with the detection antibody and then washed with PBST (0.05% PBST). The signal was detected using Pierce's SuperSignal ELISA peak chemiluminescent substrate according to the manufacturer's instructions. Luminescence was measured using a Molecular Devices M5 plate reader with an integration time of 500 ms which captures all wavelengths. The data were background corrected and the antibody concentration luminescence signal dependence was adjusted using the Langmuir isothermal equation using the GraFit program. At times when the antigen concentration was similar to the dissociation constant, the quadratic tight binding equation was used. The reported dissociation values were obtained from the adjustments to these equations; where PL represents the signal of the bound complex, B max is that maximum binding, K D is the dissociation constant and L is the concentration of the ligand.

Langmuir isothermal equation

Isothermal equation of Langmuir:

Tight bonding equation:

The anti-LOX M64 antibody was tested in three batches and found to have a KD of 6.6 nM, 5.0 nM and 5.7 nM for Lot 3, Lot 4 and Lot 5, respectively (Figure 15) . EXAMPLE 11

Kinetic measurement of M64 antibody binding to LOX by surface plasma resonance The affinity of M64 binding was assessed by surface plasma (SPR) pessonance.

Binding affinities were measured using a ProteOn Bio-Rad instrument with thermostat at 25 ° C. Binding affinities were determined using two methods, using amine coupling; one in which the antibody was immobilized and the antigen (LOX) was added and one in which the antigen (LOX) was immobilized and antibody was added. Antibody or antigen was immobilized on a GLC chip using a 1: 1 ratio of NHS to EDC provided with the ProteOn immobilization kit. The chip was first activated with NHS / EDC a mixture and then 1 pg / ml antigen or antibody in acetate buffer pH 4.5 was flowed onto activated surface to bind. This typically produced a coupling of about 500 RU's. The surface of the activated chip was then capped with the addition of 1M ethanolamine. The bound chips were stored at 4 ° C and regenerated with 50 mM sodium hydroxide.

Dissociation constants were determined by probing the chip bound with a dilution series of antibody or antigen in PBST (0.05% tween-20). Data were acquired on all six channels available on ProteOn using a non-bound channel as a reference. The collected data were analyzed using Bio-Rad's ProteOn management software.

The M64 was found to have a KD of 7 nM (Figure 16). EXAMPLE 12 The following is a listing of sequences described throughout the specification.

DOCUMENTS REFERRED TO IN THE DESCRIPTION

This list of documents referred to by the author of the present patent application has been prepared solely for the reader's information. It is not an integral part of the European patent document. Notwithstanding careful preparation, the IEP assumes no responsibility for any errors or omissions.

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Lisbon, March 25, 2015

Claims (19)

An isolated antibody or antigen-binding fragment thereof which binds to a lysyl oxidase-type 2 (L0XL2) protein, wherein said antibody or antigen-binding fragment binds specifically to the amino acid sequence of SEQ. ID No. 6 and inhibits the enzymatic activity of the L0XL2 protein, wherein the inhibition is non-competitive. The antibody or antigen-binding fragment of the same of claim 1, wherein the antibody or fragment comprises a variable heavy chain with at least 75% amino acid sequence identity to an amino acid sequence shown as SEQ. ID No. 1. The antibody or antigen-binding fragment thereof of claim 1 or claim 2, wherein the antibody or fragment comprises a variable light chain with at least 75% amino acid sequence identity to an amino acid sequence set forth as SEQ. ID N. 0 2. The antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein said antibody or antigen-binding fragment is humanised or human. 5. The antibody or antigen-binding fragment of the same of claim 4, wherein the antibody or fragment is humanized and comprises variable heavy chain CDRs with CDR1-3 amino acid sequences of GYAFTYYLIE (SEQ ID NO: 41), VINPGSGGTNYNEKFKG (SEQ ID NO: 42) and NWMNFDY, respectively, and CDR1-3 variable light chain CDRs of amino acid sequences of RSSKSLLHSNGNTYLY (SEQ ID NO: 57), RMSNLAS (SEQ ID NO: 58) and MQH-LEYPYT (SEQ ID NO: 59), respectively. The antibody or antigen-binding fragment of the same of claim 4 or 5, wherein the antibody or fragment is humanized and has a variable heavy chain with at least 75% amino acid sequence identity to an amino acid sequence shown as SEQ. 25, 26, 27 or 28. The antibody or antigen-binding fragment of the same of claim 6, wherein the variable heavy chain comprises SEQ. ID No. 27. The antibody or antigen-binding fragment thereof of any one of claims 4-7, wherein the antibody or fragment is humanized and has a variable light chain with at least 75% amino acid sequence identity to a sequence of amino acids presented as SEQ. ID No. 30, 31 or 32. The antibody or antigen-binding fragment of the same of claim 8, wherein the variable light chain comprises SEQ. ID No. 31. A humanized antibody or antigen-binding fragment thereof according to claim 1, which competes with an antigen-binding antibody or fragment thereof comprising a variable heavy chain having an amino acid sequence shown as SEQ. ID No. 1 and a variable light chain with an amino acid sequence shown as SEQ. ID No. 2 for LOXL2 binding. The antibody or antigen-binding fragment thereof of any one of claims 1-10, wherein the antibody or fragment is labeled with a therapeutic label or diagnostic label. The antibody or antigen-binding fragment thereof of any one of claims 1-11, wherein the antibody or fragment is labeled with a detectable label. The antibody or antigen-binding fragment thereof of any one of claims 1-12, wherein the antibody or fragment is a Fv, a scFv, a Fab, an F (ab ') 2, an engineered antibody gene or a monoclonal antibody. The antibody or antigen-binding fragment thereof of any one of claims 1-13, for use in a method for reducing tumor growth, reducing metastasis, inhibiting angiogenesis, treating fibrosis, or decreasing extracellular matrix formation. The antibody or antigen-binding fragment thereof for use in a method according to claim 14, wherein said antibody or antigen-binding fragment thereof is used in combination with a second therapeutic agent. The antibody or antigen-binding fragment thereof for use in a method according to claim 15, wherein the second therapeutic agent is an antibody or a chemotherapeutic agent. 17. The antibody or antigen-binding fragment thereof for use in a method according to claim 14, wherein said fibrosis is a liver fibrosis, a pulmonary fibrosis, a renal fibrosis, a cardiac fibrosis or scleroderma. The antibody or antigen-binding fragment thereof for use in a method according to claim 17, wherein said pulmonary fibrosis is idiopathic pulmonary fibrosis (IPF), cryptogenic fibrosing alveolitis, chronic fibrosing interstitial pneumonia, interstitial lung disease (ILD ), diffuse pulmonary parenchymal disease (DPLD), emphysema, chronic obstructive pulmonary disease (COPD), or chronic asthma. The antibody or antigen-binding fragment thereof for use in a method according to claim 17, wherein said liver fibrosis is cirrhosis, chronic viral hepatitis, non-alcoholic fatty liver disease (NAFLD), alcoholic steatohepatitis (ASH ), non-alcoholic steatohepatitis (NASH), primary biliary cirrhosis (PBC), biliary cirrhosis or autoimmune hepatitis. Lisbon, March 25, 2015